Production of extracellular matrix, conditioned media and uses thereof

ABSTRACT

Provided is a matrix for promoting survival and differentiation of cells transplanted thereon, comprising a base matrix and a cell-made matrix thereon. Methods and means for making and using same are also provided. Also provided are conditioned media, related compositions, related methods, and related packaging products.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority of U.S. Provisional Application No.61/471,951 filed on Apr. 5, 2011 and U.S. Provisional Application No.61/561,224 filed on Nov. 17, 2011. This application is acontinuation-in-part of U.S. patent application Ser. No. 12/738,839,which is a 35 U.S.C. §371 National Phase application of InternationalApplication Serial No. PCT/US2008/080408, filed Oct. 19, 2008, whichclaims benefit of priority under 35 U.S.C. §119(e) to U.S. ProvisionalApplication No. 60/999,601 filed on Oct. 19, 2007. The disclosures ofthe aforementioned applications are incorporated herein by reference.

GOVERNMENTAL SUPPORT

The Research leading to the present invention was supported in part, byNational Institutes of Health Grant No. NIH RO3 EY013690. Accordingly,the U.S. Government has certain rights in this invention.

FIELD OF INVENTION

This invention relates to the production of an extracellular matrix,conditioned media, and related uses.

BACKGROUND

Disease-related changes may mask extracellular matrix ligandavailability to transplanted cells, impairing post-attachment events andleading, in turn, to cell death or inability of the cells todifferentiate. In addition, disease-related changes in the extracellularmatrix can promote cell death, leading to the clinical situation inwhich cell transplantation is contemplated.

One of the conditions in which cell transplantation may be useful isage-related macular degeneration. (In addition, other conditionsaffecting the macula, such as retinitis pigmentosa and Stargardtdisease, may benefit from cell-based therapy.) The macula lutea is anarea of the retina that is about 5000 μm in diameter. The center of themacula, the fovea, contains specialized photoreceptors and provides highacuity vision necessary for reading, driving, and recognizing faces. Inorder for light-sensing photoreceptors to function properly, they mustbe in intimate contact with a cell layer called the retinal pigmentepithelium (RPE). The photoreceptors and RPE exchange nutrients andother materials. The choroid is a vascular layer of the eye wallinterposed between the sclera and RPE, and its capillaries, termed thechoriocapillaris, provide the blood supply to the RPE andphotoreceptors. The RPE is separated from the choriocapillaris by a thinlayer of collagenous tissue called Bruch's membrane.

Age-related macular degeneration (AMD) is the most important cause ofnew cases of blindness in patients older than 55 years of age in theindustrialized world. RPE cells may be one of the targets of thepathological processes that cause AMD. Approximately 10% of patientswith AMD lose central vision. Among the ˜75% of AMD patients withcentral visual loss, abnormal blood vessels, termed choroidal newvessels (CNVs), grow from the choriocapillaris and leak fluid and bloodunder the RPE and macula (exudative or “wet” AMD), which causes visualloss. The stimulus for CNV growth in AMD is complex, and the biochemicalpathways are now being identified. One critical element is vascularendothelial growth factor (VEGF), which is involved in CNV growth andleakage. Among ˜25% of AMD patients with severe central visual loss, theRPE and foveal photoreceptors die in the absence of CNVs (atrophic or“dry” AMD, also termed geographic atrophy (GA)). No visually beneficialtreatment exits for ˜60-75% of AMD patients.

Existing therapy has significant limitations. Antioxidants, for example,do not seem to be effective in the prevention of early AMD (i.e.,drusen, retinal pigmentary changes). The Age-Related Eye Disease Study(AREDS) did not show a statistically significant benefit of the AREDSvitamin and mineral formulation for either the development of newgeographic atrophy or for involvement of the fovea in eyes withpre-existing geographic atrophy.

Pharmacological therapies (e.g., AVASTIN® and LUCENTIS®, both of whichblock the action of VEGF) that are pathway-based have provided the besttreatment results for AMD patients that have ever been reported.Nonetheless, a need for improved therapy remains. Although LUCENTIS®treatment is associated with moderate visual improvement in 25-40% ofpatients according to the results of two randomized studies, theremaining 60-75% of patients are in urgent need of an alternativeapproach. Also, these medications currently are administered viarepeated intravitreal injection, which entails some risk andinconvenience for the patient. Further, pharmacological therapygenerally involves administration of a finite number of compounds andusually involves fluctuations in drug levels above and below the desiredlevel.

Accordingly, novel methods and compositions are desired which wouldaddress these drawbacks of currently accepted treatment of AMD.

SUMMARY OF INVENTION

The instant invention addresses the drawbacks of the prior art byproviding, in one aspect, a modified base matrix for promoting survivaland/or differentiation of target cells thereon, the modified base matrixcomprising a cell-made extracellular matrix (which is a mixture ofproteins and other substances) on its surface.

In different embodiments of the invention, the step of creating thecell-made extracellular matrix may be achieved by culturing, on the basematrix, the cells capable of producing such extracellular matrix, and/orby treating the base matrix with solubilized components of theextracellular matrix and/or at least an active fraction of theconditioned media from the cells capable of producing such extracellularmatrix. Combination of these approaches is also contemplated.

In another aspect, the invention provides a method of increasingsurvival and/or differentiation of target cells on a base matrix, themethod comprising: creating a cell-made extracellular matrix on the basematrix to produce a modified base matrix and administering the targetcells to the modified base matrix. In different embodiments of theinvention, the matrices include, without limitations, those describedabove.

In another aspect, the invention provides a method of increasingsurvival and/or differentiation of target cells on a base matrix throughproviding a soluble formulation of the extracellular matrix orconditioned media to the apical surface of the cells to stimulateself-assembly and deposition of extracellular matrix and/or stimulationof mechanisms for cell survival and differentiation.

In further embodiments of the invention the base matrix may be abiological matrix, such as Bruch's membrane or a synthetic polymer basedmatrix.

The cells capable of producing the extracellular matrix are in differentembodiments selected from corneal endothelial cells, RPE cells, humanembryonic stem (ES) cells and any combinations thereof. In a preferredset of embodiments, the cells are corneal endothelial cells, including,without limitations, bovine corneal endothelial cells (BCE).

In different embodiments, the target cells suitable for the methods ofthe instant invention are selected from RPE cells, umbilical cells,placental cells, adult stem cells, human ES cells (or other embryonicstem cells), cells derived from human ES cells (e.g. RPE derived from EScells, retinal progenitor cells), fetal RPE cells, adult iris pigmentepithelial (IPE) cells, Schwann cells, and combinations thereof. Thetarget cells may be derived from an autologous or an allogeneic source.

In another aspect, the invention provides a conditioned media fromculturing the cells capable of producing the extracellular matrix. Thecells capable of forming the extracellular matrix may be the cells asdescribed above. In a preferred embodiment, the media is collected afterthe cells reach confluency.

In another aspect the invention provides an active fraction of theconditioned media, as described in the previous paragraph. The activefraction is characterized by the depletion of bioactive componentshaving molecular weight less than 20 kD, preferably less than 30 kD,more preferably, less than 50 kD, more preferably, less than 70 kD, morepreferably, less than 80 kD, more preferably, less than 90 kD, and mostpreferably, less than 100 kD. The active fraction may also be comprisedof a combination of any of the above molecular weight fractions.

In yet another aspect, the invention provides a method of treating aneye disease associated with degradation of an in situ extracellularmatrix in the eye; such treatment includes creating a modified basematrix and administering the target cells to the modified base matrix.

In different embodiments of this aspect of the invention, the modifiedbase matrix is created according to any of the embodiments of theprevious aspect of the invention. Further, the target cells are chosenas described in any of the embodiments of the previous aspects of theinvention.

In yet another aspect, the invention provides a kit for improvingsurvival and differentiation of target cells on a matrix. Generally, thekit includes at least an active fraction of the conditioned media orsolubilized extracellular matrix according to any embodiments describedherein. The kit may also include a base matrix. In another set ofembodiments, the kit comprises a modified base matrix. Further, in anyembodiments of this aspect of the invention, the target cells may beprovided.

In a further aspect, the invention provides a method for making aconditioned medium. The method includes (i) obtaining a plurality ofcells capable of forming a cell-made extracellular matrix; (ii)culturing the cells in a first medium for a period of time to form asecond medium; and (iii) collecting the second medium thereby making theconditioned medium. In one embodiment, the first medium is free ofserum. The cells can be selected from the group consisting of cornealendothelial cells, RPE cells, IPE cells, embryonic stem cells, bonemarrow-derived stem cells, placental cells, and umbilical cells.Preferably, the cells are corneal endothelial cells, such as bovinecorneal endothelial cells. The period of time can be 1-5 days, such as2-4 days or 3 days.

The invention also provides a composition comprising, consistingessentially of, or consisting of a fraction of the aforementionedconditioned medium. In one embodiment, the fraction comprises, consistsessentially of, or consists of molecules having MW of less than 3 kD. Inanother, the fraction comprises, consists essentially of, or consists ofmolecules having MW of less than 50 kD, e.g., 10-50 kD or 50 kD. In apreferred embodiment, the composition comprises, consists essentiallyof, or consists of a first fraction and a second fraction of the medium,where (i) the first fraction composition comprises, consists essentiallyof, or consists of molecules having MW of less than 3 kD, and (ii) thesecond fraction com composition comprises, consists essentially of, orconsists of molecules having MW of 10-50 kD. For example, the fractioncan comprise, consist essentially of, or consist of one or more proteinsselected from those listed in Table 6 below. The just-describedcomposition can be a pharmaceutical composition and contains apharmaceutically acceptable carrier.

The above-described composition can be used for treating age-relatedmacular degeneration (AMD). To that end, one can identify a subject inneed of such treatment and administer to the subject an effective amountof the composition.

The above-described composition can also be used as a cell culturemedium for use in storing, preserving, or inducing differentiation ofcells or tissues. Accordingly, this invention also provides a packagingproduct containing the composition, a cell or a piece of tissue, and acontainer holding the composition. The cell can be selected from thegroup consisting of retinal pigment epithelial (RPE) cells, stem cells,and corneal cells. The tissue can be selected from the group consistingof RPE derived from precursor cells, RPE derived from human embryonicstem cells, RPE derived from iPSC stem cells, whole retinae, wholecornea, tissues, and neural tissues and organs. The cell can be insuspension or in a support matrix designed for cell delivery. Thecomposition can be used at a temperature within the range of 0-40° C.,such as 4-30° C. or 15-25° C. In one embodiment, the composition and thecell or tissue can be sealed in the container.

In any embodiment of this aspect of the invention, suitable non-limitingexamples of the base matrices, the modified base matrices, and thetarget cells are those described in the other aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates that long-term survival of fetal RPE on agedsubmacular human Bruch's membrane is impaired if the surface (basementmembrane or superficial surface of the inner collagenous layer (ICL)) isnot treated.

FIG. 2 demonstrates fetal RPE resurfacing on aged human submacularBruch's membrane is improved following resurfacing with bovine cornealendothelial cell extracellular matrix (BCE-ECM).

FIG. 3 demonstrates that resurfacing aged human submacular Bruch'smembrane with a biologically deposited extracellular matrix (ECM)improves long-term RPE survival compared to untreated Bruch's membraneby over 200%.

FIG. 4 illustrates RPE survival on submacular human Bruch's membrane ofan AMD donor (age 79 years) cultured in serum-containing bovine cornealendothelial cell-conditioned media (BCE-CM) vs. routine RPE culturemedia. In the absence of Bruch's membrane treatment, these RPE cellsgenerally show poor survival on human submacular Bruch's membrane of AMDeyes after 21 days in organ culture.

FIG. 5 illustrates improved RPE survival with 21 day exposure toserum-containing BCE-CM compared to 2 day exposure on human peripheralBruch's membrane from a non-AMD donor (age 80 years).

FIG. 6 demonstrates that overnight treatment with serum-freeBCE-conditioned media results in improvement of RPE survival anddifferentiation on human submacular Bruch's membrane.

FIG. 7 demonstrates that RPE derived from human embryonic stem cellssuccessfully survive on aged human submacular Bruch's membrane treatedwith serum-free BCE-conditioned media (BCE-CM) (A, B), compared tountreated submacular Bruch's membrane (C, D).

FIG. 8 illustrates that soaking a polycaprolactone (PCL) scaffold inserum-free BCE-conditioned media (BCE-CM) results in improved initialRPE attachment (A) compared to no BCE-CM treatment (B).

FIG. 9 illustrates that fetal RPE attachment and survival at 5 days isimproved if the RPE are initially cultured in serum-free BCE-conditionedmedia for 2 days (A) vs. no BCE-CM treatment (B).

FIG. 10 illustrates that fetal RPE cultured in RPE complete media canattach and resurface an untreated PCL scaffold, but by 7 days the cellsdo not exhibit density arrest.

FIG. 11 shows a two-dimensional gel with spot identification of BCEC-CM(uncut, consisting of all fractions).

FIG. 12 provides graphical analysis of cell survival (nuclear density)on aged and AMD Bruch's membrane in molecular cut filtrates utilizing3-300 kDa filters: the nuclear densities after culture in the retentates(comprising molecules above the filter size) are compared to BCEC-CMthat has not be subject to ultrafiltration (uncut).

FIG. 13 represents results of cell survival (nuclear density) on agedand AMD Bruch's membrane in molecular cut filtrates utilizing 30-100 kDafilters. The nuclear density after culture in the filtrates is comparedto BCEC-CM that has not been subject to ultrafiltration (uncut).

FIG. 14 shows comparison of cell viability at day-3 in an RPE medium at4° C. (left) and room temperature (RT, right), where Live/dead testsperformed at day-3 showed more cell death in the RPE medium at 4° C.than at room temperature (red: ethidium homodimer staining of deadnuclei; green: calcein staining of live cells). There did not appear tobe any intact cells remaining in the 4° C. RPE medium well afterstaining.

FIG. 15 shows cell viability at day-7 in CM, CM molecular cut filtrates,and RPE medium at 4° C. (left column) and room temperature (rightcolumn); at day-7, the majority of cells remaining after live/deadstaining were dead in CM and CM fractions at 4° C. The majority of cellsin CM and CM fractions at room temperature were alive and confluent withsmall defects while only cell debris remain in the well stored in theRPE medium.

FIG. 16 shows cell viability after storage using CM batch 29AD.

FIG. 17 shows change in cell viability after storage at 4° C. using CMbatch 58.

FIG. 18 shows change in cell viability after storage at room temperatureusing CM batch 58.

FIG. 19 shows change in live cells after storage at 4° C.

FIG. 20 shows change in live cells after storage at room temperature.

FIG. 21 shows comparison of cell behavior after seeding and culture ontissue culture plastic in CM (left column) vs. RPE (right column) mediumwith media change 3×/week; the images were taken at approximately thesame location for all 3 time points.

FIG. 22 shows comparison of cell behavior after seeding and culture ontissue culture plastic in RPE medium (top row) vs. CM (bottom row) withmedia change 3×/week.

FIGS. 23A-G show paired submacular explants from a 74-year-old femalewith soft drusen, seeded with hES-RPE. CM vehicle: (A) Postmortemclinical photograph shows soft drusen (arrow) in the macula. Inset is ahigher magnification image of the area indicated by the arrow. Thedrusen are not easily visualized in this photomicrograph due to postmortem changes. (B and C) No intact cells are seen on the culturedexplant. BCEC-CM: (D) Arrow points to a patch of confluent soft drusenin the macula of the fellow eye, shown in the high magnification inset.(E) Cells almost fully resurface the explant with small defects incoverage. Cells are variable in size and shape. Insert. Cells aregenerally flat with most exhibiting short apical processes on theirsurfaces. (F and G) Cells resurfacing the explant are in a monolayer ofvery flat and elongate cells. Arrowhead (G) points to cell containingvesicles. CM vehicle nuclear density (ND), 0; BCEC-CM ND, 19.90±0.35.Scale bar: (E) 100 μm; (E, inset) 20 μm; (F) 50 μm; (G) 20 μm. Toluidineblue staining.

FIGS. 24A-G show paired explants from an 81-year-old male with nosubmacular pathology, seeded with fetal RPE. (A, D) No submacularpathology is seen in the post mortem clinical photographs. CM vehicle:(B) Cellular debris but no intact cells are seen on the surface ofBruch's membrane. Few remaining patches of RPE basement membrane(arrows, insert) are present. (C) Rare single cells are seen on theexplant surface. Arrow points to a single, very flat cell. BCEC-CM: (E)The explant is almost fully resurfaced with small defects in cellcoverage (arrows). Patches of small, rounded cells are interspersed withlocalized areas where cells are more variable in size and shape. Cellsexpress abundant short apical surface processes on their surfaces(inset). (F, G) The explant is resurfaced by a mono- and bilayer ofcells. Arrowhead (G) points to a cell overlying a cell on Bruch'smembrane. CM vehicle ND, 0.51±0.16; BCEC-CM ND, 26.8±0.41. Scale bar:(E) 100 μm; (E, inset) 20 μm; (F) 50 μm; (G) 20 μm. Toluidine bluestaining.

FIGS. 25A-G show paired explants from a 75-year-old female with largebilateral subfoveal choroidal new vessels (CNVs), seeded with fetal RPEafter mechanical CNV removal. (A, D) Arrows point to CNVs in postmortemclinical photographs; insets show CNVs after surgical dissection fromBruch's membrane. CM vehicle: (A) The CNV was approximately 4.8×3 mm.(B, C) No intact cells are seen on the explant surface. BCEC-CM: (D) TheCNV was approximately 4×3.5 mm. (E) Fetal RPE fully resurface theexplant with some areas of thick multilayers (arrowhead). The cellsurfaces are covered with apical processes (inset). (F) Cellsresurfacing the explant are predominantly monolayered with localizedareas where thin or spindle-shaped cells overlay cells on Bruch'smembrane. The cells resurfacing the explant are more variable in sizeand shape than those observed on explants from donors with geographicatrophy. (G) Fetal RPE are able to resurface small drusen (arrow pointsto druse on Bruch's membrane) and basal laminar deposits (asterisk).Arrowhead points to a cell with a darkly staining irregular-shapednucleus. CM vehicle ND, 0; BCEC-CM ND, 25.10±0.30. Scale bar: (E) 100μm; (E, inset) 20 μm; (F) 50 μm; (G) 20 μm. Toluidine blue staining.

FIGS. 26A-G show paired explants from an 82-year-old female withgeographic atrophy, seeded with fetal RPE. The patient's clinicalhistory noted AMD for 20 years. (A, D) Postmortem clinical photographsshowing subfoveal geographic atrophy before RPE seeding. CM vehicle: (B)Only a few dead cells (arrows) and cellular debris are present on theexplant surface. (C) No cells are present on the Bruch's membranesurface. BCEC-CM: (E) RPE fully resurface Bruch's membrane in the areaof geographic atrophy with a few very small defects (arrows). Localizedareas of multilayering are present. Cell surfaces show abundant apicalprocesses (inset). (F) In this field, cells resurfacing the BCEC-CMexplant are predominantly bilayered. Cells directly on Bruch's membraneare small and tightly packed; flat cells appear to overlie the cells incontact with Bruch's membrane. (G) Flattened cell processes overlyingcells on top of Bruch's membrane are indicated by arrowheads. The cellprocesses contain vesicles. CM vehicle ND, 0; BCEC-CM ND, 19.61±0.43.Scale bar: (E) 100 μm; (E, inset) 20 μm; (F) 50 μm; (G) 20 μm. Toluidineblue staining.

FIGS. 27A-G show paired explants from an 80-year-old male withintermediate-size drusen in the CM vehicle-cultured eye and intermediateand large drusen in the BCEC-CM-cultured eye, seeded with cultured adultRPE (isolated from a 70-year-old donor). CM vehicle: (A) Two drusen(closely clustered) were present in the macula (arrow and arrowhead,high magnification inset). The drusen are not easily visualized in thesephotomicrographs due to post mortem changes. (B) Very few large cellsare observed on the explant surface (6 cells in this image field). Arrowpoints to a pair of very large, flat cells. (C) No cells are present onthe surface of Bruch's membrane. BCEC-CM: (D) A cluster of drusen (arrowand high magnification inset) can be seen in the macula of the felloweye. (E) The explant is fully resurfaced by a monolayer of cells thatare highly variable in size. Some of the cells within the monolayer donot have intact cell membranes (cells that appear white in the lowmagnification image), and some cells have died with remnants of cellulardebris (arrows). The high magnification inset shows that most of thecells are covered with short apical processes, including cells that arevery large (fetal RPE that are of this size on submacular Bruch'smembrane generally have smooth surfaces with no apical processes). Onecell in the field exhibits surface blebs. (F, G) Cells resurfacing theexplant are generally large and often pigmented. Localized areas ofbilayering are present (F, arrowheads). CM vehicle ND, 0.14±0.14;BCEC-CM ND, 12.0±0.77. Scale bar: (E) 100 μm; (E, inset) 20 μm; (F) 50μm; (G) 20 μm. Toluidine blue staining.

FIGS. 28A-B show nuclear densities of cells seeded on aged submacularBruch's membrane explants after 21-day culture in conditioned mediumvehicle (CM vehicle) or BCEC-conditioned medium (BCEC-CM) (pairedexplants from the same donor). (A) Nuclear density comparison of RPEderived from human embryonic stem cells (hES-RPE, N=6), cultured humanfetal RPE (fRPE, N=22), and cultured human adult RPE (RPE donor ages 58,71, 78 years; N=7). Within each group, significant differences wereobserved between cells cultured in CM vehicle and cells cultured inBCEC-CM. The nuclear density of cells cultured in CM vehicle was notstatistically different between groups. The nuclear densities of hES-RPEand fRPE were not significantly different from each other but weresignificantly higher than the nuclear density of adult RPE after culturein BCEC-CM. (B) Comparison of nuclear densities of fRPE on age-matched,non-AMD vs. AMD Bruch's membrane at day-21. Explants seeded with fRPE onaged Bruch's membrane (N=9) were compared to explants seeded on AMDsubmacular Bruch's membrane (N=13). No significant differences wereobserved in the nuclear densities of fRPE on non-AMD vs. AMD explantsfor a given medium although the nuclear density was significantly higherin the presence of BCEC-CM vs. CM vehicle. Nuclear density values arecounts of nuclei of cells directly in contact with Bruch's membrane,expressed as mean nuclear density/mm Bruch's membrane. Bars are meannuclear density±standard error. *P<0.05; **P<0.001.

FIG. 29 shows nuclear densities of fetal RPE cultured in BCEC-CM for 3-(N=7), 7- (N=8) or 14-days (N=6) followed by culture in CM vehicle for atotal culturing period of 21 days. Submacular Bruch's membrane explantsfrom fellow eyes were cultured in BCEC-CM for the entire 21-day period.Nuclear densities were significantly higher when cultured for the entire21-day period in BCEC-CM compared to shorter periods of time in BCEC-CM.Nuclear density after three-day culture was significantly lower thannuclear density after 14-day culture in BCEC-CM. Bars are mean nucleardensity±standard error. *P<0.05.

FIG. 30 shows comparison of fetal RPE nuclear density after 21-dayculture in different media and on different surfaces. Nuclear densitiesof fetal RPE after culture in BCEC-CM on aged and AMD Bruch's membrane(N=43), BCEC-ECM-resurfaced aged Bruch's membrane cultured in RPE medium(N=11), and young Bruch's membrane cultured in RPE medium (N=5) were notsignificantly different. Culture on Bruch's membrane from aged and earlyAMD donors in RPE medium (N=33) resulted in significantly lower nucleardensities than that observed in BCEC-CM cultured, BCEC-ECM-resurfaced,and young Bruch's membrane explants. RPE nuclear density after culturein CM vehicle on aged and AMD Bruch's membrane (N=22) was significantlylower than culture in RPE medium on aged and early AMD explants. BCEC-CMexplant nuclear densities are combined data from 21-day fetal RPEnuclear density counts of the Effects of BCEC-CM on Long-Term CellSurvival study (FIG. 28A) and 21-day BCEC-CM controls from the CellSurvival Following Different BCEC-CM Culture Times Study (FIG. 29). Datafor BCEC-ECM resurfaced explants and young donor explants are fromSugino et al. Invest Ophthalmol Vis Sci 2011; 52:1345-1358. data forfetal RPE on aged (including early AMD) Bruch's membrane explants werecombined data from Sugino et al. Invest Ophthalmol Vis Sci 2011;52:4979-4997 and Sugino et al. Invest Ophthalmol Vis Sci 2011;52:1345-1358 (data were not significantly different, P=0.745). Bars aremean nuclear density±standard error. *P<0.05.

FIGS. 31A-F show fetal RPE ECM deposition onto tissue culture dishesafter 7-, 14-, and 21-day culture in BCEC-CM or RPE medium. ECM isdeposited to a higher degree when cells are cultured in BCEC-CM (A-C)over the 21-day period compared to that observed after culture in RPEmedium (D-F). Increase in the numbers of thick fibers can be seen inBCEC-CM culture with time while thick fiber deposition seems to be lessextensive at all time points after culture in RPE medium. ECM coating ofthe tissue culture plastic is evident by the disappearance of theculture plastic striations (barely discernable in BCEC-CM cultures atday-7) at day-14 and -21. In RPE medium, culture plastic striations canbe seen at day-7 and -14 but not at day-21, indicating that somematerial coats the culture dish. Scale bar, 50 μm; 0.1% Ponceau S stain,phase contrast.

FIGS. 32A-L show immunocytochemical labeling (epifluorescence) ofcollagen IV, laminin, and fibronectin deposition onto tissue culturedishes after 7-day culture in BCEC-CM or RPE medium. BCEC-CM: CollagenIV labeling (A) is visualized as a network of fibers with some thickenedfibers and localized areas of continuous coating. Laminin labeling (B,E) is similar of that of collagen IV although not as extensive. Lamininappears to colocalized with some collagen IV fibers (C, collagen IV,laminin overlay). Fibronectin labeling (D) is an open network of fiberswith some areas of where fibers appear to have heavier deposition.Localized non-fibrous coating of the tissue culture dish can be seenadjacent to fibers. Fibronectin-laminin overlay (F) shows someco-localization of label. RPE medium: Collagen IV (A) labeling is moreextensive than laminin (H, K). Very little fibronectin labeling (J) ispresent. Some co-localization of collagen and laminin is seen in theoverlay (I). Labeling of all three ECM proteins is not as extensive asthat seen after BCEC-CM culture (images for each protein photographed atsame exposures). Scale bar, 200 μm.

FIGS. 33A-L show immunocytochemical labeling (epifluorescence) ofcollagen IV, laminin, and fibronectin deposition onto tissue culturedishes after 14-day culture in BCEC-CM or RPE medium. BCEC-CM: CollagenIV (A), laminin (B, E) and fibronectin (D) deposition is more extensivethan that seen at day-7 (FIG. 32). All three proteins show extensiveresurfacing of the tissue culture dish with small defects in coverage.Collagen IV and laminin are highly co-localized (C) while fibronectinand laminin are co-localized in part (F). RPE medium: Collagen IV (G)and laminin (H, K) labeling are more extensive than at day-7 (FIG. 32)but are not as extensive as labeling seen after culture in BCEC-CM forthe same time period. Very little fibronectin (J) is present. Images foreach protein in the two conditions were photographed at the sameexposure. Collagen IV and laminin are extensively co-localized (I) whilefibronectin and laminin (L) are co-localized in part. (Intensity offibronectin labeling has been increased for the overlay.) Scale bar, 200μm.

FIGS. 34A-L show immunocytochemical labeling (epifluorescence) ofcollagen IV, laminin, and fibronectin deposition onto tissue culturedishes after 21-day culture in BCEC-CM or RPE medium. BCEC-CM: Similarto 14-day culture, collagen IV (A) and laminin (B, E) extensivelyresurface the culture dish and are highly co-localized (C). Fibronectin(D) does not appear to be as extensively deposited as collagen IV andlaminin and is colocalized in part with laminin (F). RPE medium:Collagen IV (G) and laminin (H, K) appear to deposited at levels similarto those seen at day-14 and are not as extensive as that deposited afterculture in BCEC-CM. Both proteins appear to be co-localized (I). Verylittle fibronectin was detected (J). Images for each protein in the twoconditions were photographed at the same exposure. Scale bar, 200 μm.

FIGS. 35A-M show ECM deposition under fetal RPE on Bruch's membrane froma 70-year-old donor (no submacular pathology) after 21-day culture inBCEC-CM or RPE medium. BCEC-CM: (A) Calcein imaging of cells on Bruch'smembrane prior to removal with ammonium hydroxide. The explant isresurfaced almost completely with small, highly fluorescent cells. Smalldefects are present in the RPE layer. (B) SEM of the surface of Bruch'smembrane revealed after cell removal in an area where the ECM has beendamaged (possibly at the time of cell removal or during confocal imagingmanipulation), demonstrating the difference in surface morphology of theinner collagenous layer (ICL) vs. the newly deposited ECM. Arrows pointto the folded edge of the ECM. (C) A network of open and fused fiberscovered the surface of the ICL. The ECM forms a fairly continuous sheetin some areas. High magnification inset shows the ECM surface details.(D-F) Both collagen IV (D) and laminin (E) covered the explant with anextensive mesh-like deposition. There was some colocalization of label(F, overlay). (G) Control (no primary antibody, overlay) imaged atsimilar pinhole settings as (D) and (E) with higher detector gain inboth FITC and rhodamine channels. Very little fluorescence is seen ineither channel with some choroidal autofluorescence (FITC) seen in theupper left (arrow) of the image (tissue was not flat). (H-J) Fibronectinlabeling (H) of ECM fibers was evident on the explant while lamininlabeling (I), similar to that seen in (E), was seen in fibers, betweenfibers, and as punctate labeling associated with fibers. (Punctatelaminin labeling shows up best in the overlay (J)). Fibronectin andlaminin did not appear to be co-localized to any significant degree (J,overlay). (K) Control (no primary or secondary antibodies, overlay)imaged at the same settings as H-J. Only faint autofluorescence could bedetected. RPE medium: (L) Calcein imaging of the explant shows cellsresurfaced Bruch's membrane with several small defects in the RPE layer(arrows point to small RPE defects in the submacular area and a largedefect on one edge (asterisk). Photographed at the same intensitysettings as (A), the overall intensity of calcein imaging appears to beless than on (A) except at the edge of the large defect. (M) SEMexamination of the surface of this explant revealed no ECM depositionconfirming negative labeling (not shown) of all three markers byconfocal examination. Collagen fibers are partially obscured bydeposits. (A, L) epifluorescence; (B, C, M) SEM; (D-K) confocalcompressed z-stacks. SEM scale bar, 10 μm; inset (C), 5 μm. Confocalscale bar, 50 μm.

DETAILED DESCRIPTION

In order to alleviate the drawbacks of the prior art, cell-based therapymay offer advantages over pharmacological therapy. Cell-based therapy toreplace lost or diseased RPE has the potential to preserve and restorevision in: 1) age-related macular degeneration (AMD) patients withevolving atrophy and/or choroidal neovascularization, 2) patientssuffering from traumatic RPE-Bruch's membrane injury, and 3) patientswith other diseases associated with RPE dysfunction (e.g., Stargardtdisease and some forms of retinitis pigmentosa). In addition toreplacing lost or diseased RPE with cells capable of performing RPEfunctions, transplanted RPE may be able to rescue nearby dyingphotoreceptors through their known capacity to secrete substances suchas neurotrophic factors and cytokines.

As noted above, pharmacological therapy involves administration of afinite number of compounds and usually involves fluctuations in druglevels above and below the desired level. In contrast, cells placed insitu express a plethora of molecules (e.g., neurotrophic factors,cytokines) that can inhibit pathological processes and rescue neuronsthat are damaged by disease. Moreover, they can express these moleculesin amounts, combinations, and frequencies that are tailored precisely tomolecular changes that occur from moment to moment. Thus, cells have thecapacity to function as “factories” that produce many more substances atappropriate doses and times than can be managed with conventionalpharmacological therapy. This pharmacological salutary capacity ofcell-based therapy is termed “rescue”.

Another capacity of cell-based therapy is “replacement,” which refers tothe ability of transplanted cells to replace native cells that havedied. In diseases such as AMD, RPE and photoreceptor cell deathconstitutes a component of “irreversible” visual loss in many patients.Among AMD patients with evolving atrophy, RPE transplantation could becurative.

The first efforts to develop cell-based therapy for AMD involved RPEtransplantation after CNV excision. Before current pharmacologicaltherapy was available, CNV excision was proposed as a treatment forCNVs. In most AMD patients, CNV excision is associated with iatrogenicRPE defects due to the intimate association of RPE cells and the CNV.Combined RPE transplantation and CNV excision has been attempted in AMDeyes, but it has not yet led to significant visual improvement in mostpatients. In contrast, RPE transplantation in animal models of retinaldegeneration has been proved to rescue photoreceptors and preservevisual acuity. Although animal studies validate cell transplantation asa means of achieving photoreceptor rescue, an important distinctionbetween humans with AMD and laboratory animals in which RPEtransplantation has been successful is the age-related modification ofBruch's membrane in human eyes, which may have a significant effect onRPE graft survival.

With normal aging, human Bruch's membrane, especially in the submacularregion, undergoes numerous changes (e.g., increased thickness,deposition of extracellular matrix (ECM) and lipids, cross-linking ofprotein, non-enzymatic formation of advanced glycation end products).These changes and additional changes due to AMD could decrease thebioavailability of ECM ligands (e.g., laminin, fibronectin, and collagenIV) and cause the poor survival of RPE cells in eyes with AMD. Thus,although human RPE cells express the integrins needed to attach to theseECM molecules, long-term transplanted RPE cell survival on agedsubmacular human Bruch's membrane is impaired.

Because the changes in Bruch's membrane from aging and AMD are complexand may not be fully reversible, one approach is to establish a new ECMover Bruch's membrane. Adding exogenous ECM ligands (e.g., combinationsof laminin, fibronectin, vitronectin, and collagen IV) can improve RPEattachment to aged Bruch's membrane to a limited degree. (Del Piore etal., Curr Eye Res. 2002; 25:79-89). These results are consistent withthe hypotheses that ECM ligand availability may decrease with Bruch'smembrane aging and that it is possible to increase ligand density onthis surface.

It is doubtful that attention to individual ECM ligands withoutattention to their 3-dimensional organization will be highly effective(as indicated by the results of previous studies). The instantdisclosure demonstrates that bovine corneal endothelial cells (BCE) canattach to Bruch's membrane and, more importantly, lay down ECM. Thus,Bruch's membrane can be resurfaced with a complex ECM that is known tosupport excellent RPE growth and differentiation and that iswell-defined biologically (Tseng et al., J Biol. Chem. 1981; 256:3361-5;Gospodarowitz et al, J Cell Physiol. 1983; 114:191-202; Robinson et al.,J Cell Physiol. 1983; 117:368-76; Nevo et al., Connect Tissue Res. 1984;13:45-57; Sawada et al., Exp Cell Res. 1987; 171:94-109; Kay et al.,Invest Ophthalmol Vis Sci. 1988; 29:200-7).

The inventors have surprisingly found that RPE focal adhesion formationon aged submacular Bruch's membrane is abnormal compared to that seen onBCE-ECM-coated culture dishes. Without wishing to be bound by anyparticular theory, the inventors hypothesized that this early event,probably resulting from poor ECM ligand availability, underlies laterdegenerative changes in RPE cells on aged Bruch's membrane after theyattach. RPE focal adhesion formation is markedly improved onBCE-ECM-coated aged submacular Bruch's membrane six hours after seeding.RPE cells seeded onto the BCE-ECM-coated Bruch's membrane uniformlyresurface the submacular explants with small, compact cells of variableshape. As discussed in the examples, the inventors' data demonstratethat resurfacing by BCE-ECM enhances RPE cell long-term survival on agedsubmacular human Bruch's membrane by ˜230% (see FIG. 2A-C and FIG. 3),which is in marked contrast to previous studies in which only modestimprovement was seen following treatment with soluble ECM ligands. RPElong-term survival and differentiation are enhanced via this approach.

The research described in the instant application has demonstrated thatsurvival of transplanted cells depends critically on the surface onwhich the transplanted cells grow. In two animal models, allogeneic RPEtransplants can survive for at least short periods of time in thesubretinal space and that freshly harvested RPE sheets ormicroaggregates are similarly successful. (Wang et al., InvestOphthalmol Vis Sci. 2001; 42:2990-9; Wang et al., Exp Eye Res. 2004;78:53-65). In pigs, there is more inflammation associated with freshlyharvested sheets than with cultured dispersed cell transplants, possiblydue to the greater trauma associated with sheet transplantation.

AMD-related changes as well as iatrogenic changes associated withchoroidal new vessel (CNV) excision create a damaged Bruch's membranesurface in eyes undergoing CNV excision. (Nasir et al., Brit. J.Ophthalmol. 1997; 81:481-9; Zarbin, Arch Ophthalmol. 2004; 122:598-614).In most cases, surgical damage to Bruch's membrane includes removal ofthe RPE basement membrane and removal of portions of the innercollagenous layer (ICL). Aged adult RPE can resurface RPE defects onaged submacular human Bruch's membrane in organ culture only to alimited extent. (Wang et al., Invest Ophthalmol Vis Sci. 2003;44:2199-2210) In addition to the surface affecting the ability of agedadult RPE to resurface RPE defects, aged RPE per se are impaired intheir ability to attach and grow in culture and on Bruch's membrane.(Tsukahara et al., Exp Eye Res 2002; 74(2):255-266; Zarbin. Trans AmOphthalmol Soc 2003; 101:499-519; Wang, et al., J Rehabil Res Dev 2006;43: 713-22; Ishida, et al., Curr Eye Res. 1998; 17: 392-402).

Resurfacing is even more limited if RPE migration/ingrowth must occur onthe ICL. The in vitro wound healing data accurately predict the outcomein AMD patients following CNV removal, who show incomplete ingrowth ofRPE with associated photoreceptor degeneration. (Hsu et al., Retina.1995; 15:43-52). Freshly harvested, aged adult RPE cells, such as wouldbe used in autologous transplants, do not survive on aged submacularBruchs membrane. (Tsukahara et al., Exp Eye Res. 2002; 74:255-66).Culturing adult RPE cells upregulates integrins necessary for cellattachment. (Zarbin, Trans Am Ophthalmol Soc. 2003; 101:499-520).However, aged adult RPE never grow as robustly on aged Bruch's membraneas young RPE. Histopathology of an AMD eye that underwent unculturedadult RPE transplantation confirms these predictions. (Del Priore etal., Am J. Ophthalmol. 2001; 131:472-80).

Long-term studies of cultured fetal human RPE on aged submacular humanBruch's membrane show that many cells do not survive, and if they arepresent, they do not appear to be adequately differentiated. (Gullapalliet al., Exp Eye Res. 2005; 80:235-48). Of the various cell types studiedto date (including adult stem cells, embryonic stem cells(differentiated into RPE-like cells), adult and fetal RPE, and adultiris pigment epithelial (IPE) cells), none appear to survive anddifferentiate adequately on aged submacular human Bruch's membrane.(Zarbin et al., 2003; Gullapalli et al., 2005; Gullapalli et al., TransAm Ophthalmol Soc. 2004; 102:123-37; discussion 137-8; Itaya et al.,Invest Ophthalmol Vis Sci. 2004; 45:4520-8).

Thus, in one aspect, the invention is drawn to a modified base matrixfor survival and/or differentiation of RPE cells thereon, the modifiedbase matrix comprising a cell-made extracellular matrix thereon.

In different embodiments, the base matrices suitable for the instantinvention may be protein-based matrices, including, without limitations,collagen (including gelatin), solubilized human basement membrane, andfibrinogen-based formulations. These synthetic matrices can includemixtures optimized according to concentration of base formulations andadditional cell-supporting molecules added to said formulations.

In other embodiments, the base matrices may comprise non-proteinaceouspolymers, such as, for example, polycaprolactone (PCL), polylactic acid(PLA), polyglycolic acid (PGA), poly(lactide-co-glycolide) (PLGA),poly(methyl methacrylate) (PMMA), polyorthoester matrices, and anycombinations thereof.

In yet another set of embodiments, the base matrices may be biologicalmembranes, such as, for example a Bruch's membrane. In one embodiment,the Bruch's membrane used as a base matrix of the instant invention isan aged Bruch's membrane. The term “aged” essentially depends on aspecies source of the membrane used (e.g., assuming that the source ofthe membrane is human, the membrane over 40 years old, or 50 years old,or 60 years old, or 70 years old, or 80 years old, or 90 years old, or100 years old). The species source of the Bruch's membrane include,without limitations primates, e.g., gorilla, chimpanzee, orangutan, andhuman. If the source of the membrane is not human, the age of themembrane should be adjusted accordingly, based on the life span of thesource species.

The matrices described and/or exemplified in any of the embodiments ofthe invention may be located in vivo or in vitro.

The methods of production of the base templates depend on the nature ofthe template. For example, if the template is polymer-based (e.g., PCLbased), it may be chemically synthesized. If the template is abiological membrane, as described above, it can be surgically harvestedand cultured according to the methods known in the art, including,without limitations, those described in the Examples below.

Once the base matrix is chosen and obtained, it is modified with anextracellular cell-made matrix to produce a modified base matrix. Thesuitable cells capable of forming matrices are well known in the art andinclude, without limitations corneal endothelial cells (including, butnot limited to, bovine cells), RPE cells (including, but not limited to,human), IPE cells (including, but not limited to, human), and stem cells(including, but not limited to, human embryonic stem cells, placentalstem cells, umbilical stem cells, bone marrow-derived stem cells, neuralprogenitor cells).

The choice of the cells capable of forming the extracellular cell-madematrices ultimately depends on the nature of target cells that are to begrown on the modified base matrix. In a set of embodiments, wherein thetarget cells that are grown on the modified base matrix are RPE, cornealendothelial cells, e.g., bovine corneal endothelial cells (BCE) presenta suitable option.

Another aspect of the invention is the application of conditioned media.It may be applied in one of three ways: 1) as a modification of the basematrix, 2) as a solution or in a biocompatible and degradable matrixapplied to the apical surface of transplanted cells, or 3) as part ofthe vehicle in which the cells are transplanted.

The methods of culturing BCE cells are well known in the art althoughspecifics of the methods may vary slightly (see, e.g., Bonanno et al.,Am J Physiol Cell Physiol 277: C545-C553, 1999; Tseng et al., J. CellBiol 1983; 97:803-809; Katz, et al, Invest Ophthal Vis Sci 1994;35:495-502; Gospodarowicz, et al., Exp Eye Res 1977; 1:75-89; MacCallumet al., Exp Cell Res 1982; 139:1-13; Vlodaysky Curr Protocols Cell Biol1999; 10.4.1-10.4.14. Briefly, according to the protocol published byBonanno, the primary cultures from fresh cow eyes are established inT-25 flasks with 3 ml of DMEM, 10% bovine calf serum, and anantibiotic-antimycotic (100 U/ml penicillin, 100 μg/ml streptomycin, and0.25 μg/ml Fungizone); gassed with 5% CO₂-95% air at 37° C., and mediachanged every 2-3 days. These are subcultured to three T-25 flasks andgrown to confluence in 5-7 days. The resulting second-passage culturesare then subcultured onto coverslips or filters, reaching confluencewithin 5-7 days.

Another method of culturing BCE is to establish freshly isolated cellson tissue culture dishes (diameter 35, 60, or 100 mm) in Dulbecco'smodified Eagle's medium (DMEM) supplemented with RPE complete media(DMEM with 2 mM glutamine, 15% fetal bovine serum, 2.5 μg/ml fungizone,0.05 mg/ml gentamicin, 1 ng/ml basic fibroblast growth factor (bFGF)).Cells are grown in a humidified incubator at 10% CO₂-95% air at 37° C.until confluent with media change every 2-3 days. Upon confluency, cellsare passaged at a split ratio of ˜1:3.7. First passage cells are grownin RPE complete media until confluent; second passage cells aregenerated by passaging first passage cells at a split ratio of ˜1:7.3.

ECM can be generated by culturing (including but not limited to) first,second, or fourth passage cells in ECM media (DMEM with 2 mM glutamine,10% fetal bovine serum, 5% donor calf serum, 2.5 μg/ml fungizone, 0.05mg/ml gentamicin, 1 ng/ml bFGF, 4% dextran). 1 ng bFGF is added every2-3 days until cells are confluent. ECM can be harvested from cells atconfluence or up to 3 months post-confluency. Time of ECM harvesting isspecific to the cell depositing ECM. (BCE require less time to depositECM than RPE, including RPE derived from human ES.) Cells can be removedfor ECM harvesting by exposure to 0.02M NH₄OH and/or PBS and/ordetergents (e.g., 0.5% triton X-100) and/or urea (2M).

Conditioned media is generated by growing cells following passage inmaintenance media (ECM media without dextran). bFGF may or may not beadded every 2-3 days. 48-72 hours prior to collection, cells are washeda minimum of 3× in DMEM with no supplements to remove serum. Media iscollected after 48-72 hour culturing in MDBK-MM or other base medium.

In different embodiments of the invention, the modified base matrix maygenerally be created by at least three techniques: first, thematrix-forming cells are cultured on the base matrix; second, the matrixis deposited by cells onto culture dishes and harvested; and third, thematrix-forming cells are cultured separately from the base matrix, andthe tissue culture media from the matrix-forming cells is collected.Harvested deposited ECM and/or media from culture may be administered tothe base matrix, may be applied to the apical surface of cells, or maybe used as a vehicle for cell transplantation. Apical application of theECM and/or conditioned media can be by one of the following methods(including but not limited to): injection of the ECM and/or conditionedmedia solely or in a biocompatible, biodegradable matrix and/orinjection following transplant cell attachment or placement onto Bruch'smembrane; incorporated into the overlying material (e.g., gelatin) usedfor transplanting cell sheets or embedded single cells or cellaggregates. The combination of these techniques is also contemplated.

If the first or second option is employed, the matrix-formed cells maybe stripped from the base matrix by chemical methods, such as, forexample, NH₄OH or Urea or detergent wash or PBS soaking. Enzymaticmethods (e.g., trypsin digestion) are less desirable due to possibleprotein damage.

If the third option is employed, it is important to keep in mind thatserum, which may be present in the conditioned media, usually containsligands of the cell-made (extracellular) matrix in the media.Accordingly, the suitable media should preferably be serum free, or atthe very least, serum depleted to reduce the likelihood of inducing aninflammatory/immune response in the transplant recipient.

After sufficient time, e.g., at least 7 days or at least until culturesreach 100% confluency, or at least 1 week after confluency, or at least2 weeks after confluency, or at least 3 months after confluency) themodified base matrix is formed to a degree sufficient to improvesurvival and differentiation of the cells which are to be grown on themodified base matrix (i.e., target cells). In other words, thesufficient time may be less than 3 months, or less than 2 weekspost-confluency, or less than 1 week post-confluency, or less than 7days. As discussed above, the target cells may include, withoutlimitations, RPE, umbilical cells, placental cells, adult stem cells, EScells, bone marrow-derived stem cells, fetal RPEs, adult iris pigmentepithelial (IPE) cells, neural progenitor cells, Schwann cells, and anycombination thereof, and may be derived from an autologous or anallogeneic source.

In another aspect, the invention provides a method of increasingsurvival and/or differentiation of target cells on the base matrix, themethod comprising: creating cell-made extracellular matrix on said basematrix to produce a modified base matrix and administering to saidmodified base matrix said target cells.

According to this aspect, the base matrix and the modified base matrixinclude, without limitations, the base matrices and the modified basematrices as described according to the previous aspect of the inventionor as disclosed in the examples below.

The target cells include, without limitations, the target cellsdescribed above. In one embodiment, the cells are RPE. The RPE cells maybe chosen or differentiated from multiple sources. For example, RPE maybe differentiated from stem cells, such as embryonic or adult stemcells, or RPE may be fetal RPE. The methods of in vitro differentiationof RPE are known in the art.

For example, if one desires to differentiate the RPE from ES cells, USPublication 20070196919 discloses a suitable exemplary method for doingso. Briefly, the H-1 (WA-01) human embryonic stem cell line may beobtained from a commercial or a non-commercial source, such as WicellResearch Institute. The cells are cultured and passaged on a feederlayer made of irradiated mouse embryonic fibroblasts. Embryoid bodiesare formed by treating undifferentiated hES colonies with 1 mg/ml oftype IV collagenase (Invitrogen) and resuspending them in a 6-wellultra-low attachment plate (VWR) in the presence of media containingDMEM:F12 (Gibco), 10% knockout serum (Invitrogen), B-27 supplement(Invitrogen), 1 ng/ml mouse noggin (R&D Systems), 1 ng/ml humanrecombinant Dkk-1 (R&D Systems), and 5 ng/ml human recombinantinsulin-like growth factor-1 (IGF-1) (R&D Systems). The cells arecultured as embryoid bodies for 3 days. On the fourth day, the embryoidbodies are plated onto poly-D-lysine-Matrigel (Collabora-tive Research,Inc)-coated plates and cultured in the presence of DMEM: F12, B-27supplement, N-2 Supplement (Invitrogen), 10 ng/ml mouse noggin, 10 ng/mlhuman recombinant Dkk-1, 10 ng/ml human recombinant IGF-1, and 5 ng/mlhuman recombinant basic fibroblast growth factor (bFGF) (R&D Systems).The media is changed every 2-3 days.

Adult cells may also be used for creating RPE cells. For instance,retinal and corneal stem cells themselves may be utilized for cellreplacement therapy in the eye. In addition, neural stem cells from thehippocampus have been reported to integrate with the host retina,adopting certain neural and glial characteristics (see review of Lund,R. L. et al., 2003, J. Leukocyte Biol. 74: 151-160). Neural stem cellsprepared from fetal rat cortex were shown to differentiate along an RPEcell pathway following transplantation into the adult rat subretinalspace (Enzmann, V. et al., 2003, Investig. Ophthalmol. Visual Sci. 44:5417-5422). Bone marrow stem cells have been reported to differentiateinto retinal neural cells and photoreceptors following transplantationinto host retinas (Tomita, M. et al., 2002, Stem Cells 20: 279-283;Kicic, A. et al., 2003, J. Neurosci. 23: 7742-7749). An ocular surfacereconstruction in a rabbit model system, utilizing cultured mucosalepithelial stem cells, has also been reported.

In other embodiments, other cell types may be used for the methods ofthe instant invention. For example, US Publication 20050037491 (the '491publication) reports that placental or umbilical cells injected into aneye of a dystrophic RCS rat differentiate into cells exhibiting at leastsome RPE characteristics, as assessed by ERG recording, rod and coneresponses, a- and b-wave recording, histological examination, and Nisslstaining.

In the experiments of the '491 publication, cultures of human adultumbilical and placental cells (passage-10) were expanded for 1 passage.All cells were initially seeded at 5,000 cells/cm² on gelatin-coated T75flasks in Growth Medium. For subsequent passages, all cells were treatedas follows. After trypsinization, viable cells were counted after trypanblue staining. Briefly, 50 microliters of cell suspension was combinedwith 50 microliters of 0.04% w/v trypan blue (Sigma, St. Louis Mo.), andthe viable cell number, was estimated using a hemocytometer. Cells weretrypsinized and washed three times in supplement free-DMEM:Low glucosemedium (Invitrogen, Carlsbad, Calif.). Cultures of human umbilicalplacental and fibroblast cells at passage-11 were trypsinized and washedtwice in Leibovitz's L-15 medium (Invitrogen, Carlsbad, Calif.). For thetransplantation procedure, dystrophic RCS rats were anesthetized withxylazine-ketamine (1 mg/kg i.p. of the following mixture: 2.5 mlxylazine at 20 mg/ml, 5 ml ketamine at 100 mg/ml, and 0.5 ml distilledwater), and their heads secured by a nose bar. Cells devoid of serumwere resuspended (2×10⁵ cells per injection) in 2 microliters ofLeibovitz, L-15 medium (Invitrogen, Carlsbad, Calif.) and transplantedusing a fine glass pipette (internal diameter 75-150 micrometers)trans-sclerally. Cells were delivered into the dorso-temporal subretinalspace of anesthetized 3-week old dystrophic-pigmented RCS rats (totalN=10/cell type).

As discussed throughout this disclosure, treatment of the base matrixwith a conditioned media from BCE cells is sufficient for improvedsurvival and/or differentiation of the target cells. Accordingly, inanother aspect, the invention provides a conditioned media from culturedcells capable of producing the cell-made matrix, according to anyembodiment, as described above. In addition, the inventors havesurprisingly discovered that experiments with media harvested frompassage-2 cultures show that media harvested from cells that have beenin culture for 2 weeks after reaching confluency is not as supportive asmedia harvested at earlier time points (50% confluent, confluent, 1 weekafter confluency). Thus, in a preferred embodiment, the conditionedculture medium is harvested from the cells that have not been confluentfor more than 2 weeks.

The inventors have also discovered that the whole conditioned media isnot necessary for the improved survival and/or differentiation of theRPE on the modified base matrix. Thus, in another aspect, the inventionis drawn to the active fraction of the conditioned culture media,according to any of the embodiments described above. Specifically, theinventors have found that high molecular weight components aresufficient for the initial beneficial effect of the conditioned culturemedia. Specifically, such an active fraction may be characterized byhaving its low molecular weight components depleted. However, lowmolecular weight components may be important in long-term survival anddifferentiation.

In different embodiments, the active fraction is characterized by thedepletion of bioactive components having molecular weight less than 20kD, preferably less than 30 kD, more preferably, less than 50 kD, morepreferably, less than 70 kD, more preferably, less than 80 kD, morepreferably, less than 90 kD, and most preferably, less than 100 kD. Theactive fraction may be characterized by any combination of componentsseparated according to size or other methods (e.g., high pressure liquidchromatography (HPLC)).

The depletion of low molecular weight or other non-essential componentsmay be achieved by many methods, including, without limitation,filtration, size fractionation by gel filtration or gradientcentrifugation, HPLC (separation according to charge, size, orhydrophobicity), immunoprecipitation, affinity column separation, andthe like. However, it is important that the methods of depletion oflow-molecular weight compounds should not result in protein cleavage norshould it disrupt secondary and tertiary protein structures of anyneeded components in the medium.

While it is possible to surgically remove CNVs, CNV excision isassociated with iatrogenic RPE defects due to the intimate associationof RPE cells and the CNV. (Thomas et al., Am J Ophthalmol 1991; 111:1-7;Nasir et al., Br J Ophthalmol 1997; 81:481-489; Castellarin et al.,Retina 1998; 18:143-149; Hsu et al., Retina 1995; 15:43-52; Rosa et al.,Arch Ophthalmol 1996; 114:480-487). Combined RPE transplantation and CNVexcision has been attempted in AMD eyes, but it has not led tosignificant visual improvement in most patients. (Algvere et al.,Graefes Arch Clin Exp Ophthalmol 1994; 232:707-716; Del Priore et al.,Am J Ophthalmol 2001; 131:472-480; Binder et al., Am J Ophthalmol 2002;133:215-225; Tezel et al., Am J Ophthalmol 2007; 143:584-595; Joussen etal., Am J Ophthalmol 2006; 142:17-30). Potential causes of RPEtransplant failure in human patients include immune rejection, inabilityof transplanted RPE cells to survive and differentiate on agedsubmacular Bruch's membrane, and choriocapillaris atrophy, all causingdeath of the RPE graft. In contrast, RPE transplants rescuephotoreceptors and preserve visual acuity in animal models of retinaldegeneration. (Li et al., Exp Eye Res 1988; 47:911-917; Coffey et al.,Exp Neurol 1997; 146:1-9; Lund et al., Proc Natl Acad Sci USA 2001;98:9942-9947; Wang et al., Invest Ophthalmol Vis Sci 2008; 49:416-421;Gias et al., The European journal of neuroscience 2007; 25:1940-1948).

An important distinction between humans with AMD and laboratory animalsis the age-related modification of Bruch's membrane that occurs in humaneyes. With normal aging, human Bruch's membrane, especially in thesubmacular region, undergoes numerous changes (e.g., increasedthickness, deposition of extracellular matrix (ECM) and lipids,cross-linking of protein, nonenzymatic formation of advanced glycationend products). (Guymer et al., Prog Retin Eye Res 1999; Marshall et al.,The Retinal Pigment Epithelium. New York: Oxford University Press;1998:669-692; 18:59-90; Abdelsalam et al., Surv Ophthalmol 1999;44:1-29). Pauleikhoff and coworkers reported an age-related decline inthe presence of laminin, fibronectin, and collagen IV in the RPEbasement membrane. (Pauleikhoff et al., Ophthalmologe 2000; 97:243-250).It is possible that changes in submacular Bruch's membrane permeabilityand choriocapillary density may contribute to age-related RPE death.However, it was found that RPE survival is also impaired on agedsubmacular Bruch's membrane explants in organ culture, where diffusionof nutrients is not a factor in cell survival. This finding suggeststhat there are additional factors within aged Bruch's membrane itselfthat adversely affect RPE survival and that modification of Bruch'smembrane may have a significant effect on RPE graft survival in patientswith AMD. (Gullapalli et al., Exp Eye Res 2002; 74:255-266).

As discussed above and shown in the examples below, in the instantinvention, the use of modified base matrix according to any embodimentof the invention promotes survival and/or differentiation of cellstransplanted onto this matrix. In embodiments where the base matrix isan aged Bruch's membrane or a Bruch's membrane from an eye undergoingmacular degeneration, survival and/or differentiation of transplantedRPE was improved when the Bruch's membrane was modified withextracellular matrix from BCE cells. Importantly, the experiments wereperformed in human eyes, thus validating the methods and compounds ofthe instant invention for human treatment.

Accordingly, in one aspect, the methods according to the instantinventions may be performed for treatment of humans suffering from AMD(whether the wet AMD or the dry AMD). As mentioned above, retinaldegenerative diseases constitute the leading causes of blindness in theindustrialized world. AMD, the most prevalent of these, can be treatedpharmacologically, although at this time the majority of patients do notrecover lost vision. RPE cells may be a primary target of thepathological processes that cause AMD.

The goal of cell-based therapy as a treatment for AMD patients is toreplace diseased or dying RPE cells, which provide metabolic support forthe photoreceptors. RPE transplantation could prevent further visionloss and might even, in some cases, lead to vision improvement inselected AMD patients. Cell-based therapy in AMD patients has notreached its full potential due to the failure of cells to survive andbecome functional in the diseased AMD eye.

As disclose herein, the present invention provides a biologicallysynthesized mixture (bovine corneal endothelial cell-conditioned media,BCEC-CM) that improves transplanted RPE survival by more than 10-foldwhen tested in an organ culture bioassay utilizing aged and AMD humandonor eyes. It was found that BCEC-CM significantly improved cellsurvival on aged and AMD Bruch's membrane (the surface on which cellsmust survival in patients) using fetal RPE, aged adult RPE, and RPEgenerated from human embryonic stem cells. Identification of thebioactive molecules in BCEC-CM allows for the development of an adjunctto cell transplantation therapy in patients with AMD to ensuresuccessful cell transplant integration and functionality.

In one embodiment, the present invention provides BCEC-CM andconditioned media comprising bioactive molecules derived therefrom fortherapeutic use in AMD. Specifically, it provides fractions of BCEC-CMidentified via molecular cut filtration possessing bioactivity, one inthe <3 kDa filtrate and one found in a 10-50 kDa fraction. Thesefractions, in combination, ensure cell survival on Bruch's membrane. Thebioactive molecule or molecules in each of these fractions in BCEC-CMcan serve as an adjunct to cell transplantation therapy in patients withAMD.

In other embodiments, the methods of treatment comprise modifyingBruch's membrane with the cell made extracellular matrix, according toany embodiments described herein, and wherein the Bruch's membrane islocated in vivo. Essentially, in different embodiments, Bruch's membraneis modified when the at least the active fraction of the conditionedmedia (or the whole conditioned media) of any of the embodimentsdescribed above or exemplified below can be applied basally as asubstrate to coat the surface of Bruch's membrane, in a mixture withcells, or apically in a biocompatible matrix.

It is also worth noting that this invention has been shown to supportadult and embryonic stem cells and retinal pigment epithelial cells(adult and fetal) on human Bruch's membrane, including Bruch's membranefrom AMD eyes. Accordingly, in different embodiments, different types ofcells may be applied within the methods of this aspect of the invention.

The compositions containing the extracellular matrix (e.g., at least theactive fraction of the conditioned media according to any embodiment ofthe instant invention) can be applied to Bruch's membrane in livingpatients through a variety of strategies, e.g., direct application tothe subretinal space.

In another embodiment, the scaffold (i.e., the base matrix), such as,for example, a polymeric scaffold such as PCL, can be delivered into thesubretinal space. In different embodiments, the scaffold is modifiedwith the extracellular matrix (resulting in the modified base matrix),as described above and exemplified below. Further, such modified basematrix may be delivered in combination with a scaffold that containscells to be transplanted to the patient's eye. The suitable cells havebeen described above.

As used herein, the terms “treat” or “treatment” or “treating” etc.,refer to executing a protocol in an effort to alleviate signs orsymptoms of a disease in a subject. Alleviation may occur either beforeor after appearance of these signs or symptoms. In addition, these termsdo not require a complete alleviation of the signs or symptoms, do notrequire a cure, and include protocols resulting in only marginal effectson a patient.

A “subject” refers to a human and a non-human animal. Examples of anon-human animal include all vertebrates, e.g., mammals, such asnon-human primates (particularly higher primates), dog, rodent (e.g.,mouse or rat), guinea pig, cat, and non-mammals, such as birds,amphibians, reptiles, etc. In a preferred embodiment, the subject is ahuman. In another embodiment, the subject is an experimental animal oranimal suitable as a disease model.

In another aspect, the invention provides a kit for treatment of AMD(both wet AMD and dry AMD). Generally, the kit would include a set ofinstructions and at least the active fraction of the conditioned media,as described in any of the embodiments of the instant invention, and maycomprise the unfractionated conditioned media, also, according to any ofthe embodiments of the instant invention. Alternatively, the kit maycomprise the cells capable of producing the cell-made extracellularmatrix, according to any of the embodiments of the instant invention.Specifically and without limitations, the cells capable of producing thecell-made extracellular matrix include BCE cells. Alternatively, the kitmay include ECM generated and harvested from cell-deposited matrices insolubilized or non-solubilized form. Optionally, the kit may provide thebase matrix, according to the embodiments described above. The basematrix may be a natural polymer (e.g., a protein-based base matrix), asynthetic polymer (e.g., PCL), a biological membrane (e.g., Bruch'smembrane), or a combination thereof.

In another set of embodiments, the kit may comprise a modified basematrix, according to any of the embodiments described herein. In any ofthe embodiments of the kit, suitable target cells may also be provided,according to any of the embodiments described above.

The set of instructions may be provided in any media, including, withoutlimitations, written, graphic, audio recording, video recording, andelectronic media.

In yet another aspect, the invention provides a novel medium for andmethod of storing and differentiating cells for transplantation.Cell-based therapy represents a promising approach, which may besight-preserving and/or restoring for patients with these diseases.However, cell-based therapies are complicated by the necessary step ofgrowing, differentiating and storing cells for transplantation. Giventhe severe consequences of AMD and the often irreversible vision losswhich may occur, there exists a need for specifically-designed andimproved methods of preparing cells for transplantation.

The present invention provides a novel medium for and method ofdifferentiating, storing and preserving cells for transplantation. Themedium was developed as a measure to improve the viability andfunctionality of cells or tissues, prior to transplantation. It can beused with various types of cells and tissues, including, but not limitedto, ocular cells, ocular tissue and neural tissue. One application ofthe medium is the storage and shipment from cell manufacturer to an enduser (e.g., surgeon). The medium can further serve as a cell culturemedium, to induce rapid differentiation of RPE cells, as well as othercells and tissues. Accordingly, this invention provides a novel mediumfor storage and differentiation which provides the significant advantageof improving the health and functionality of cells, tissues or organs,at the time of transplantation.

In one embodiment, the present invention provides a storage andpreservation medium for use prior to transplantation of variousbiological transplants, including, but not limited to, cells, retinalpigment epithelial (RPE) cells, RPE derived from various cells, RPEderived from human embryonic stem cells, RPE derived from iPSC stemcells for neurodegenerative diseases, corneal cells, whole retinae,whole cornea, tissues, neural tissues and organs. This medium willsupport RPE viability during long-term storage, without mediareplacement. Furthermore, the cells can be in suspension or in a supportmatrix designed for cell delivery.

In another embodiment, the present invention provides a cell culturemedium to induce and maintain rapid differentiation of retinal pigmentepithelial cells. In a further embodiment, the present inventionprovides a cell culture medium, for RPE, as well as other cells, whichallows and assures rapid attachment onto untreated surfaces, including,but not limited to, tissue culture plastic without coating forattachment (e.g., laminin, fibronectin, and matrigel).

As disclosed herein, the medium is based on secreted molecules generatedas conditioned media (CM) containing bovine corneal endothelial cell(BCEC) secreted molecules, either the CM itself, components of the CM ora combination of components based on the identification of activemolecules in BCEC-conditioned media. The medium of present invention canbe used as a storage/preservation medium without changing orreplenishing. This medium can also be used as a cell culture medium forinducing rapid differentiation in RPE when changed three times per week.

The present invention offers distinct advantages over technologycurrently in existence. Storage and preservation media currently existfor corneas but there is no medium specifically developed for use withRPE or for other similar types of cells. The composition andconcentration of the components of the present invention varysignificantly from that of existing cornea storage media. Furthermore,when used as a differentiation medium, the present invention features arapid induction of differentiation, which exceeds that of standard RPEculture media. Morphological indicators of differentiation in fetal RPEdemonstrate that the onset of differentiation occurs very rapidly (e.g.,within one week when seeded at 3164 cells/mm²), and the cells achieve alevel of differentiation that is only observed (if at all) in long-termfetal RPE culture in standard RPE culture media.

Finally, when used as a cell culture medium for RPE, a BCEC-conditionedmedium induces unexpected rapid attachment of cells onto untreatedtissue culture plastic (attachment as soon as one hour followingseeding) and effects accelerated growth and differentiation. On theother hand, a standard RPE culture medium attachment takes approximately24 hours on untreated tissue culture plastic, with a marked differencein the rate of growth. This aspect of the present invention, a reductionof the time required to achieve differentiated RPE (or other cells,tissues or organs), provides a substantial advantage in the field ofmanufacturing. For example, in the case of induced pluripotent stemcells (iPSC), BCEC-conditioned media can reduce the time a patient hasto wait for autologous cell transplantation.

The role of the medium of the present invention as a storage mediumpresents a marked improvement over existing technology. In order tosolidify its usefulness, quantitative analysis of cell viability atdifferent time points can be performed, in order to compare cell deathrates in different media. Additionally, maintenance of RPE markers canbe compared in different media with time in culture. Since anothermethod of cell introduction at the time of transplantation issingle-cell suspensions, it can also be determined, as part of thepresent invention, whether RPE can maintain viability in suspension inBCEC-CM. Generally, RPE are anchorage-dependent cells that undergoapoptosis if not attached to a suitable substrate. Since BCEC-CMcontains many soluble ECM ligands, this medium can likely support cellsin suspension. Injection of fresh, as opposed to frozen, cells could beadvantageous for cell transplantation of since frozen cells must recoverafter thaw and tend to attach and grow sluggishly compared to freshcells. Additionally, frozen cells must be washed to remove DMSO (infreezing solution) while fresh cells could be injected directly from thestorage vial.

The present invention aims to identify the cell-supporting components inBCEC-CM and to manufacture a solution comprised of small molecules andhuman recombinant proteins for commercial development. Although an RPEmedium appears to support cells to a similar degree as BCEC-CM (exceptat the condition noted previously), the presence of fetal bovine serumin the medium is not ideal (xenogeneic proteins). The storage solutionfor commercial development can be a newly-developed product with aunique formation or can be molecules added to Optisol to increaseeffectiveness.

Since the presence of mRNA does not necessarily predict proteinpresence, the present invention can include a determination of theexpression of RPE differentiation markers (proteins) with time inculture. Long-term cultures of fetal RPE on BCEC-ECM and on tissueculture plastic in standard RPE media and BCEC-CM can be compared.Preliminary data indicate that protein expression of latedifferentiation markers (RPE65 and bestrophin) cannot be detected in theconditions tested to date (3 weeks). If fetal RPE differentiates morerapidly in BCEC-CM than in standard RPE media, similar studies onhES-RPE (Advanced Cell Technology) can be performed.

The invention will now be described in the following non-limitingexamples.

EXAMPLES Example 1 Long-Term Survival of Fetal RPE on Aged SubmacularHuman Bruch's Membrane is Impaired

Fetal RPE (3164 cells/mm²) were seeded on aged human submacular Bruch'smembrane debrided to expose the superficial surface of the innercollagenous layer. To create surfaces exposing the RPE basementmembrane, RPE were gently wiped off the RPE/choroid/sclera explant usinga wet surgical sponge. To create surfaces exposing the surface of theinner collagenous layer beneath the RPE basement membrane (i.e.,superficial ICL), following RPE removal as indicated previously, amoistened surgical sponge was use to abrade the RPE basement membrane.In general, the area of RPE basement membrane debridement was created byapproximately 5 wipes of the moistened sponge in each of 4 directions(rotating the explant 90 degrees after each series of 5 wipes). (V. K.Gullapalli, et al., Exp Eye Res 2005; 80(2):235-248). Cells were seededonto the sclera/choroid explant and cultured for 21 days and evaluatedfor resurfacing with scanning electron microscopy (SEM) and lightmicroscopy (LM). Nuclear density counts (mean±SD) of fetal RPE on agedsubmacular human Bruch's membrane at day-1 (basement membrane, N=7;superficial ICL, N=7), day-7 (basement membrane, N=6; superficial ICLN=6), day-14 (basement membrane N=7), day-21 (superficial ICL, N=6) wereperformed on 5 non-adjacent slides in the central 3 mm of the section(includes the submacular region of Bruch's membrane). Cells on tissueculture dishes coated with BCE-ECM (N=1) are included for comparison.Cells were seeded at a density of 3164 cells/mm² for all time points andsurfaces. Fetal RPE survival on submacular Bruch's membrane decreasedwith time, regardless of the surface on which the cells are seeded(e.g., RPE basement membrane or the surface of the inner collagenouslayer (superficial ICL)) (See FIG. 1, modified from V. K. Gullapalli, etal., Exp Eye Res 2005; 80(2):235-248.) (Transplanted RPE will encountersuperficial ICL in situ if native RPE are removed by CNV excision.) Incontrast, density increased to 45 nuclei/mm² if RPE are grown on bovinecorneal endothelial cell extracellular matrix (BCE-ECM)-coated culturedishes.

Example 2 Fetal RPE Resurfacing on Aged Bruch's Membrane Resurfaced withBovine Corneal Endothelial Matrix (BCE-ECM)

BCE (3164 cells/mm²) were cultured on the inner collagenous layer ofaged human submacular Bruch's membrane (65 yr. old donor) for 14 days toallow ECM deposition. Cells were culture in the same way as cellscultured for ECM deposition on culture dishes (see paragraph 0056).Following BCE removal with NH₄OH to expose the newly deposited ECM andextensive washing with PBS, explants were seeded with fetal RPE (3164cells/mm²) and cultured for 21 days. The results of these experimentsare illustrated in FIG. 2.

FIG. 2A is a scanning electron micrograph (SEM), showing that fetal RPEfully resurfaced the treated explant with large, flat polymorphic cells.Cells showed varying amounts of short apical processes on their surfaces(insert). Mag. bar 50 μm; insert mag bar 10 μm.

FIGS. 2B and 2C are light micrographs (LMs). As shown in FIG. 2B, cellsfully resurfaced the treated explant and are in a monolayer. Mag. bar100 μm. FIG. 2C is a higher magnification of the explant shown in FIG.2B, allowing one to discern the variable morphology of the cells. Cellsare tightly adherent to the explant surface. Arrow in FIG. 2C points tothe nucleus of a cell in the monolayer; arrowhead to a choriocapillarisvessel. Mag. bar 20 μm.

As a negative control, submacular Bruch's membrane of the fellow eye wasincubated in serum-free media with no BCE for 14 days followed byexposure to NH₄OH, rinsing with PBS and fetal RPE seeding and culturingfor 21 days.

FIG. 2D is a SEM of the RPE on the untreated Bruch's membrane surface.Notably, fetal RPE incompletely resurfaced the untreated innercollagenous layer. Islands of large, flattened cells are present(arrows). Dead, dying, or poorly attached cells are also present on thesurface or attached to the flattened cells (arrowhead). Asterisk,exposed inner collagenous layer surface. Mag. bar 50 μm.

FIGS. 2 E and 2F show a representative view of the RPE on untreatedBruch's membrane. In this section, there is only a single clump of cells(arrow). Mag. bar 100 μm. FIG. 2F is a high magnification of the clumpof cells shown in FIG. 2E. Arrow points to a cell in the clump that isnot intact. Arrowhead points to a choriocapillaris vessel. Mag. bar 20μm.

Example 3 Resurfacing Bruch's Membrane with a Biologically DepositedExtracellular Matrix (ECM) Improves Cell Survival

Bovine corneal endothelial cells (BCE, passage-2) were seeded onto humansubmacular superficial ICL of Caucasian donors over 55 years old at adensity of 3164 cells/mm² and cultured for 14 days to allow ECMdeposition or treated for 14 days with serum-free media only. FollowingBCE removal with NH₄OH and extensive rinsing, fetal RPE (passage-2-5)were seeded at the same density onto the treated Bruch's membranesurface and cultured for 21 days. The fellow eye was treated similarlyexcept no BCE were seeded.

RPE seeding density was 3164 cells/mm² for 21-day incubations todetermine long-term survival and morphology. FIG. 3 shows the cumulativedata from 9 explant pairs. Counts are mean fetal RPE nuclei/mm Bruch'smembrane (±SEM).

A statistically significant 230% (p=0.006) increase in cell density isseen at day-21 on treated explants compared to explants treated withserum-free DMEM only (FIG. 3) or explants in which cells were seededdirectly on Bruch's membrane with no prior treatment (FIG. 1, day-21superficial ICL (striped bar)).

Example 4 Bovine Corneal Endothelial Cells (BCE) Secrete ECM Componentsinto the Overlying Media

During ECM formation, in addition to basal secretion, BCE secrete ECMcomponents into the media (BCE-conditioned media, BCE-CM), and thecomposition and relative amounts of the components vary with culturetime and passage number. Secretion of ECM components into the overlyingmedia is most abundant in early passage cells (up to passage-2) andexceeds basal ECM deposition in quantity. (Tseng et al. J Biol Chem1981; 256:3361-3365).

Serum-free BCE-conditioned media (BCE-CM) was prepared from passage-2cells that were cultured in serum-free Dulbecco's modified Eagle'smedium (DMEM) for 48 hrs. An initial sample concentrated using a 30 kDcut-off filter identified 20 proteins by MS/MS-MALDI. The proteins in anadditional sample of conditioned media, unfiltered, were subjected to 2DLC-MS/MS, and samples were analyzed with MALDI-TOF and QTOF.

These analyses identified 84 proteins (at least one peptide having C.I.values of >95%). Conditioned media from the same preparation was alsoanalyzed by 2D gel separation, and selected spots (142) were analyzed byMALDI-TOF. This analysis identified 45 different proteins. A combinedtotal of 109 proteins were identified using these methods (Table 1).

TABLE 1 Protein components of two different samples of bovine cornealendothelial cell conditioned media (BCE-CM) as determined byMS/MS-MALDI, LC-MS/MS (MALDI-TOF and Q-TOF), and MS/MS-MALDI of selected2D gel spots. Name Protein Description IGF-1 prepro-insulin-like growthfactor I IGFBP-2 Insulin-like growth factor-binding protein-2 IGFBP-4insulin-like growth factor-binding protein-4 IGFBP-7 PREDICTED: similarto insulin-like growth factor binding protein 7 (predicted), partial FNFibronectin (FN) hypothetical hypothetical protein LOC504471 protein Hfactor 1 H factor 1 (complement) C3 Chain B, Structure Of Mammalian C3With An Intact Thioester At 3a Resolution C3 Complement component 3 C3dComplement component C3d PREDICTED: similar to Complement C3 precursor,partial Complement C3 precursor [Contains: Complement C3 beta chain;Complement C3 alpha chain; C3a anaphyl C4 PREDICTED: similar toComplement C4 precursor CCP modules CCP modules 3-12, with parts of CCP2 and 13 collagen, type I collagen, type I, alpha 2 collagen, type IIIcollagen, alpha-1 (III) chain collagen, type V type V preprocollagenalpha 2 chain collagenase type collagenase type IV precursor IV TIMP2tissue inhibitor of metalloproteinase 2 MMP2 matrix metalloproteinase 2EGF-containing PREDICTED: similar to EGF-containing fibulin-likeextracellular matrix protein 1 fibulin-like isoform a precursorextracellular matrix protein 1 PREDICTED: similar to EGF-containingfibulin-like extracellular matrix protein 1 isoform b fibulin-3, FIBL-3EGF-containing fibulin-like extracellular matrix protein 1 precursorfibulin-1 fibulin-1 C SPARC protein SPARC protein Osteonectin secretedprotein, acidic, cysteine-rich (osteonectin) ESM-1 PREDICTED: similar toEndothelial cell-specific molecule 1 precursor (ESM-1 secretory protein)apolipoprotein A apolipoprotein A-I precursor apolipoprotein Eapolipoprotein E apolipoprotein E precursor EC-SOD PREDICTED: similar toExtracellular superoxide dismutase [Cu—Zn] precursor (EC-SOD) isoform 2PCSK9 PREDICTED: similar to proprotein convertase subtilisin/kexin type1 inhibitor precursor alpha-actin alpha-actin Alpha-cardiac PREDICTED:similar to Actin, alpha cardiac (Alpha-cardiac actin) isoform 1 actinBeta-actin Actin, cytoplasmic 1 (Beta-actin) actinin PREDICTED: similarto actinin alpha 4 isoform 3 actinin, alpha 1 POTE-2 PREDICTED: similarto Prostate, ovary, testis expressed protein on chromosome 2 Dkk-3PREDICTED: similar to Dickkopf related protein-3 precursor (Dkk-3)(Dickkopf-3) (hDkk-3) dickkopf homolog 3 cathepsin L cathepsin LFibulin-1 PREDICTED: similar to Fibulin-1 precursor isoform 1Thrombospondin- Chain A, Crystal Structure Of The Thrombospondin-1N-Terminal Domain 1 Vimentin Vimentin PGDS prostaglandin D2 synthaseprecursor ITI PREDICTED: similar to inter-alpha trypsin inhibitor heavychain precursor 5 isoform 1 nidogen nidogen Entactin PREDICTED: similarto Nidogen precursor (Entactin) isoform 3 Osteonidogen PREDICTED:similar to Nidogen-2 precursor (NID-2) (Osteonidogen) MGP matrix Glaprotein HSPG PREDICTED: heparan sulfate proteoglycan 2 heparan sulfateproteoglycan perlecan PREDICTED: similar to Basement membrane-specificheparan sulfate proteoglycan core protein precursor nephronectinPREDICTED: similar to nephronectin isoform b FSTL1 Follistatin-relatedprotein 1 precursor (Follistatin-like 1) (TGF-beta-inducible proteinTSC-36) FSTL3 PREDICTED: similar to Follistatin-related protein 3precursor (Follistatin-like 3) (Follistatin-rel LTBP-2Latent-transforming growth factor beta-binding protein 2 precursor(LTBP-2) albumin albumin transthyretin transthyretin NPC Alveolarmacrophage chemotactic factor (Neutrophil chemotactic protein) (NPC)CTGF connective tissue growth factor Dimeric Bovine Chain B, DimericBovine Tissue-Extracted Decorin, Crystal Form 2 Tissue-Extracted DecorinGOLPH2 PREDICTED: similar to golgi phosphoprotein 2 KIAA1133 PREDICTED:similar to KIAA1133 protein GST glutathione S-transferase, GST{N-terminal} {EC 2.5.1.18} [cattle, erythrocytes, Peptide Partial, 2serine protease serine protease SMC1 mitosis-specific chromosomesegregation protein SMC1 homolog superfast myosin PREDICTED: similar tosuperfast myosin heavy chain AEBP1 AE binding protein 1 angiomodulinangiomodulin anti-PPS anti-pneumococcal capsular polysaccharideimmunoglobulin heavy chain variable region N-cadherin Cadherin-2precursor (Neural-cadherin) (N-cadherin) (CD325 antigen) CD44 CD44antigen precursor (Phagocytic glycoprotein I) (PGP-1) (HUTCH-I)(Extracellular matrix receptor APT Chain A, Crystal Structure Of TheFirst Active Autolysate Form Of The Porcine Alpha Trypsin ACTHpreproadrenocorticotropic hormone (ACTH) corticotropincorticotropin-like interm lobe peptide cystatin C cystatin C (amyloidangiopathy and cerebral hemorrhage) [Bos taurus] fibromodulinfibromodulin galanin galanin polypeptide precursor polypeptide (AA −31to 1139) PTGDS prostaglandin H2 D-isomerase microglobulin similar tobeta 2-microglobulin lumican lumican gelsolin gelsolin cadherin 11cadherin 11, type 2 preproprotein Transferrin Serotransferrin precursor(Transferrin) (Siderophilin) (Beta-1- metal-binding globulin) PDIProtein disulfide-isomerase A3 precursor (Disulfide isomerase ER-60)(ERP60) Nucleobindin 1 Nucleobindin 1 Calnuc Chain A, Nmr SolutionStructure Of The Calcium-Binding Domain of Nucleobindin (Calnuc)Osteonectin Secreted protein, acidic, cysteine-rich Beta-globinHemoglobin subunit beta (Hemoglobin beta chain) (Beta-globin) Alphaenolase Alpha enolase FHOS2S splicing FHOS2S splicing variant variant [Aldehyde aldehyde dehydrogenase family 1, subfamily A1 dehydrogenaseLDHA Lactate dehydrogenase-A ATIC-ALK Tropomyosin 4-anaplastic lymphomakinase fusion protein Tyrosine 3-/tryptophan 5-monooxygenase activationprotein, epsilon polypeptide Human Annexin Chain A, Structure Of HumanAnnexin A2 In The Presence Of Calcium Ions A2 Peroxiredoxin 1Peroxiredoxin 1 Peroxiredoxin 6 Peroxiredoxin 6 Anti-oxidantAnti-oxidant protein 2 (non-selenium glutathione peroxidase, acidiccalcium- protein 2 independent phospholipa) NME4 Expressed innon-metastatic cells 1 protein biglycan biglycan clusterin clusterinosteoglycin plasminogen activator inhibitor type 1, member 2transketolase Proteins were identified based on at least one peptidewith C.I. value 95% or more.

Example 5 Bovine Corneal Endothelial Cell Conditioned Media (BCE-CM) CanImprove RPE Cell Survival on Aged Human Submacular Bruch's Membrane

BCE-CM containing serum was prepared by exposing newly confluentcultures of BCE to RPE complete media (DMEM with 2 mM glutamine, 15%fetal bovine serum, 2.5 μg/ml fungizone, 0.05 mg/ml gentamicin, 1 nag/mlbFGF) for 3 days. Media was centrifuged and supernatant stored frozen.Submacular aged human Bruch's membrane explants were debrided to exposethe superficial inner collagenous layer; 3164 cells/mm² were seeded oneach explant and cultured for 21 days. Explants with cells were culturedin serum-containing BCE-CM or RPE complete media.

Preliminary data using serum-containing BCE-CM as media for RPEfollowing seeding onto peripheral (N=2) and submacular Bruch's membrane(N=1, with submacular drusen) shows BCE-CM used as media supports betterRPE attachment and long-term survival than RPE complete media (FIG. 4).In the experiments illustrated in FIG. 4, submacular human Bruch'smembrane of an AMD donor (age 79 years) was treated withserum-containing bovine corneal endothelial cell-conditioned media(BCE-CM) vs. routine RPE culture media; BCE-CM was prepared by exposingpassage-2 BCE for 3 days in RPE complete media containing serum. Theexplant to be treated had a greater number of large submacular drusenthan the control explant, which means it was the more severely diseasedof the two eyes. A. RPE cultured in BCE-CM, day 21. Cells fullyresurface the treated explant with a few small defects (arrow). Highmagnification insert shows short apical processes on the surface of somecells and along cell borders. B. RPE cultured in RPE complete media,day-21. The explant is sparsely resurfaced with patches or clumps ofRPE. Original magnifications 200×; insert 1000×.

The inventors have also shown serum-containing BCE-CM used as mediaduring the duration of the incubation (21 days) showed better cellmorphology and resurfacing on peripheral inner collagenous layer ofBruch's membrane than explants where BCE-CM was changed to standard RPEmedia (which also contains serum) after 2 days (FIG. 5). In theseexperiments, RPE survival on peripheral Bruch's membrane from a non-AMDdonor (age 80 years) was investigated. A. Aged human peripheral Bruch'smembrane explant cultured for 21 days in BCE-CM. Explant is fullyresurfaced with a fairly uniform monolayer of cells. High magnificationinsert shows short apical processes covering the surface of the cells.B. Aged human peripheral Bruch's membrane (isolated from fellow eye ofthat shown in A) explant cultured for 2 days in BCE-CM then placed inRPE complete media for 19 days. Explant is incompletely resurfaced(arrows point to defects) with cells of varying size and morphology.High magnification insert shows the cells have a few very short apicalprocesses, and the cells are fairly large and smooth. Originalmagnifications: 200×; inserts: 1000×.

Example 6 Bovine Corneal Endothelial Cell Conditioned Media (BCE-CM)Supports Rapid RPE Attachment and Spreading on Non-Tissue CultureTreated Plastic to a Similar Degree as Cells on BCE-ECM-Coated TissueCulture Plastic

Soluble ECM can affect cell shape and metabolism in addition tostimulating production of ECM molecules. The inventors performed studiesto determine: 1) whether soluble components in BCE-CM can be usedinstead of BCE-ECM to coat culture dishes and support fetal RPE growthand differentiation; and 2) whether BCE-CM used as media for cellsuspension and seeding can support cells on non-tissue culture treateddishes (NTC). Since serum contains ECM ligands (e.g., vitronectin andfibronectin), these studies were performed in serum-free media as themost stringent test of cell support. Because of RPE dependence on serumin the media for long-term survival, experiments were performed for 3days only.

Serum-free conditioned media (sfBCE-CM) was prepared from passage-2cultures as described above. sfBCE-CM was applied in media or by coatingnon-tissue culture treated dishes (NTC) unconcentrated or inconcentrated form (8-fold, using a 30 kD cut-off filter). Negativecontrol was cells seeded and cultured in DMEM only. Fetal RPE(passage-3) were seeded at a density of 526 cells/mm² for all attachmentstudies. To determine whether non-protein components of BCE-CMcontribute to early attachment and spreading of fetal RPE, sfBCE-CM washeated to 80° for 15 minutes, centrifuged, and the supernatant was usedas media for attachment and seeding of fetal RPE. The importance ofintact protein components in BCE-CM evidenced by cell behavior inheat-treated sfBCE-CM was confirmed by treatment with proteinase Kagarose beads (removed prior to cell suspension and seeding) before andafter heat treatment.

sfBCE-CM used either as media (Table 2, A) or as a substrate to coattissue culture dishes (Table 2, B) supported rapid RPE adhesion and celldivision in serum-free conditions. sfBCE-CM-treated dishes supportedrapid attachment and spreading by 1 hour (Table 2, B), similar toBCE-ECM-treated dishes (Table 2, D). Fetal RPE seeded in heatinactivated and/or proteinase K-treated BCE-CM behaved similar to thoseon NTC (Table 2A, C). Cells seeded onto 8× sfBCE-CM did attach andspread but to a slightly lesser degree than on unconcentrated CM. Thebest morphology (uniform spreading, less filopodia formation) wasobserved in cells on BCE-ECM and in sfBCE-CM used as media or as acoating substrate. Experiments are in progress to determine whetherdifferences in cell behavior are observed in media harvested from BCE ofdifferent passages and times in culture.

TABLE 2 Fetal RPE behavior in serum-free BCE conditioned media(sfBCE-CM) under different culture conditions. Experimental Condition 1Hour Day-1 Day-3 A. RPE CELL ATTACHMENT AND GROWTH IN sfBCE-CM sfBCE-CM~40% ~70-80% spread, almost Confluent, uniform cell size, spreadconfluent few vacuoles sfBCE-CM, heat Rounded Few cells, filopodia Morecells than at day-1 but inactivated very few, poor morphology. sfBCE-CM,proteinase Rounded Rounded Rounded K treatment with and without priorheat inactivation B. sfBCE-CM SURFACE COATING OF NTC DISHES FOLLOWED BYRPE SEEDING WITH DMEM OR sfBCE-CM MEDIA sfBCE-CM, DMEM 40-50% Highdensity, confluent in Confluent, small cells, good, spread center, somemulti- morphology, few vacuoles nucleate cells sfBCE-CM, sfBCE-CM ~70%Moderate density, Elongate cells with filopodia spread filopodia andlamellipodia and lamellipodia, subconfluent 8X sfBCE-CM, ~60% Highdensity of cells, Almost confluent, more MW > 30K, DMEM spread confluentin center, similar variable morphology than 1X to 1X BCE-CM, DMEMBCE-CM, DMEM 8X sfBCE-CM, 50-60% Moderate density of cells, Elongatecells with filopodia MW > 30K, sfBCE-CM spread filopodia andlamellipodia and lamellipodia, subconfluent C. NEGATIVE CONTROL NTCplastic, DMEM Rounded Rounded Few elongated cells D. POSITIVE CONTROLBCE-ECM surface ~50-90% Majority cells are spread Cells spread andproliferating, spread but not confluent, some almost confluent filopodiaCells were seeded at the same density for all experiments. A. Effect ofsfBCE-CM as media for attachment and growth. sfBCE-CM, heat inactivatedsfBCE-CM, and sfBCE-CM treated with proteinase K are compared. B. Effectof sfBCE-CM as a surface treatment for attachment and growth onnon-tissue culture treated (NTC) dishes, either unconcentrated orconcentrated 8X using a 30 kD cut-off filter. Cells were suspended andcultured in either DMEM or sfBCE-CM. C. Control (cells on untreated NTCdishes with DMEM as media). D. Control (cells on BCE-ECM with DMEM asmedia).

Preliminary experiments with media harvested from passage-2 culturesshow that media harvested from cells that have been in culture for 2weeks after reaching confluency is not as supportive as media harvestedat earlier time points (50% confluent, confluent, 1 week afterconfluency) (data not shown). Protein composition analysis is currentlyunderway to determine changes in the media harvested at these differenttime points to determine what proteins may account for the decreasedcell support.

Example 7 Soaking in Serum Free BCE-CM can Improve Cell Survival on AgedAMD Bruch's Membrane

FIG. 6 demonstrates that even a relatively short treatment (i.e.,overnight) leads to improvement in RPE survival on and resurfacing ofBruch's membrane. In this experiment, submacular Bruch's membrane froman 80 year-old Caucasian male donor was debrided to expose thesuperficial inner collagenous layer. Large submacular drusen werepresent on Bruch's membrane of both eyes, and the eye treated withBCE-CM showed more deposits (i.e., was the more severely diseased of thetwo eyes). Bruch's membrane was treated by overnight soaking of theexplant in serum-free BCE-CM; the fellow eye explant was soaked for thesame period of time in regular serum-free media (DMEM). Fetal RPE wereseeded onto both Bruch's membrane explants at a seeding density of 3164cells/mm². Both explants were cultured in RPE complete media for 21days.

The explant treated with the conditioned media as described in theprevious paragraph shows almost 100% resurfacing with a few smalldefects (FIG. 6A, B, original magnification 200×). The highmagnification images (1000×) show small cells with varying amounts ofapical processes (a differentiation feature) (FIG. 6C, D). In contrast,the untreated explant shows incomplete resurfacing by very large flatsmooth cells (FIG. 6 E, F, original magnification 200×). Areas ofcellular debris are evident where the cells have died (arrows).Asterisks indicate areas not resurfaced.

Example 8 Treatment of Aged Bruch's Membranes with BCE Conditioned MediaImproves Survival of RPE Derived from Human ES Cells

In order to investigate whether treatment of surfaces withBCE-conditioned media improves survival and/or differentiation of RPEother than fetal RPE, the following experiments were performed. Fresh(not frozen) RPE derived from human ES cells (hES-RPE obtained fromAdvanced Cell Technology, Inc.) of intermediate pigmentation were seededonto the inner collagenous layer of submacular Bruch's membrane from a63 year-old Caucasian female at a seeding density of 3164 cells/mm².There was no evident pathology in the macula of either eye. The treatedexplant was cultured in serum-containing BCE-CM while the untreatedexplant was cultured in RPE complete media. Explants were harvestedafter 21 days in culture.

The results of these experiments are illustrated in FIG. 7. The treatedexplant (FIG. 7 A, B) demonstrated some degree of resurfacing by thehES-RPE with defects in coverage. The cells were very flat and did notshow prominent differentiated features. The untreated explant (FIG. 7C,D) showed only sparse coverage, with only a few cells in the submacularregion of the explant. Thus, BCE-CM treatment improved hES-RPE survivalon aged human Bruch's membrane.

Example 9 Active Components in BCE-CM Supporting Early Attachment andSpreading in Cell Culture

Different preparations of sfBCE-CM of different molecular weight cut-offwere prepared to determine the MW fraction of active components insfBCE-CM. Media were reconstituted to 1× following filtration. Retentatesolutions of MW>3 kD, >10 kD, >30 kD, and >50 kD and filtrate solutionsof MW<3 kD, <10 kD, <30 kD, and <50 kD were prepared. RPE (passage-4)were suspended in each solution and seeded onto non-tissue culturedtreated plastic (NTC) as detailed above. In a separate study, sfBCE-CMwas filtered using a 100 kD molecular cut off filter, yielding filtratesof <100 kD and retentates of >100 kD. Fetal RPE (passage-3) behavior wasobserved in the 2 solutions up to day-2.

The active cell-supporting components in BCE-CM appear to at leastinclude molecular weight (MW) 30 kD and higher. Based on day-3observations of vacuole formation (early apoptotic changes) in RPEcultured in retentate fractions containing proteins of molecular weightless than 30 kD, it appears that proteins present in the low molecularweight fractions may have a negative effect on the cells. Molecularweight fractions of 100 kD and higher supported rapid initial RPEattachment in serum-free media. In this assay, it was not found thatmolecular weight fractions below 100 kD supported rapid attachment andspreading in serum-free media to any degree. Yet, as will be discussedin Example 12 below, two additional bioactive fractions were identifiedthat contributed to cell survival on human submacular Bruch's membrane.

Thus, it appears that high molecular weight fractions (>100 kD) areimportant in initial RPE attachment and spreading in serum-freeconditions.

TABLE 3 Fetal RPE behavior in serum-free BCE conditioned media ofdifferent molecular weights. Molecular weight fraction of sfBCE-CM 1Hour Day-1 Day-3 Low MW (<3, 10, 30, Rounded Rounded Rounded or 50 kD)High MW (>3, 10, 30, 30-40% spread >3 kD confluent, >10, 30, Allconfluent, >3 kD smallest or 50 kD) or 50 kD almost confluent cells withmost vacuoles; >10 to confluent with kD vacuoles, uniform cellintercellular gaps size; >30 kD less vacuoles, uniform cell size; >50 kDmixed sizes, no vacuoles sfBCE-CM (unfiltered 30-40% spread Confluentwith Confluent, mixed sizes, few control for above intercellular gapsvacuoles studies) >100 kD ~70% attached ~90% spread, some Noobservations and spread filopodia (more than seen in cells on BCE-ECM)<100 kD 80-90% attached, 20-30% spread, others No observations round areround, abundant filopodia sfBCE-CM (unfiltered ~70% attached ~90%spread, some No observations control for 100 kD cut- and spreadfilopodia off studies) RPE were seeded at the same density for allexperiments. The effects of sfBCE-CM as media for attachment and growth,prepared by centrifugal filtration of different MW cut-offs (retentatesabove MW 3, 10, 30, 50, 100 kD and filtrates below MW 3, 10, 30, 50, 100kD) are shown.

Example 10 BCE Conditioned Media is Effective at Dilutions up to 20×

Fetal RPE (passage-3, 526 cells/mm²) were seeded onto non-tissue culturetreated plastic in dilutions of serum-free BCE conditioned media(sfBCE-CM, 1:1 to 1:80 dilutions) to determine the maximum effectivedilution of BCE-CM for support of initial RPE attachment and spreading.Negative control was cells seeded in serum-free DMEM. Results (Table 4).Support of attachment and spreading was seen in BCE-CM diluted up to1:10 in serum-free DMEM. Cells in 1:20 and higher dilutions showincreasingly poor attachment and morphology at day-1 after seeding.

TABLE 4 Fetal RPE behavior in diluted serum-free BCE-conditioned media.Dilution of sfBCE-CM 1 Hour Day-1 1:1 ~60-70% attached ~90-95% attachedand well spread and spread 1:5 ~60-70% attached ~90-95% attached andwell spread and spread 1:10 ~50-55% attached ~90% attached and wellspread and spread 1:20 ~50 attached ~60% attached, not as well spreadand spread as higher concentrations. Cells aggregated. 1:40 ~30-40%attached ~30-40% attached, variably spread. and spread Cells aggregated;variable morphology with lamellipodia, filopodia. Some cells elongated,some not spread. 1:80 <5% attached and <10% attached, some elongatedspread minimal spreading. Cells aggregated; all of poor morphology withelongation, filopodia and lamellipodia. DMEM Rounded, few All rounded.(negative spread control) Fetal RPE were suspended in differentdilutions of serum-free BCE-CM and seeded onto non-tissue culturetreated dishes.

Example 11 Rpe can Attach and Grow on PCL Scaffolds

1052 fetal RPE/mm² were seeded onto 5 mm diameter PCL scaffolds andcultured for 1 day. To assess attachment onto the scaffolds, cellbehavior was compared on scaffolds with no treatment (FIG. 8, B) vs.scaffold soaked in serum-free BCE conditioned media (sfBCE-CM, soakedfor ˜1 hr. at 37° C.) to allow protein adsorption (FIG. 8, A). Cellswere cultured in DMEM or in sfBCE-CM. RPE were visualized on thescaffolds with calcein imaging. RPE appeared to attach only to scaffoldstreated with or cultured in sfBCE-CM (Table 5, 1 day and FIG. 8).Greatest attachment and spreading were observed in cells seeded ontosfBCE-CM-soaked scaffolds.

To determine whether cells could eventually adhere and spread on thescaffolds, scaffolds were exposed to sfBCE-CM by either soaking (FIG. 9,A), followed by cell seeding and culturing in DMEM for 2 days or usingsfBCE-CM as media for 2 days. Controls were cells on untreated scaffoldsin DMEM for 2 days (FIG. 9, B). Cultures were changed to RPE completemedia (DMEM with 2 mM glutamine, 15% fetal bovine serum, 2.5 μg/mlfungizone, 0.05 mg/ml gentamicin, 1 ng/ml bFGF) after day 2 and culturedfor 3 days. RPE were able to resurface the scaffolds only if thescaffold was pre-soaked in sfBCE-CM or sfBCE-CM was used as media fortwo days (see FIG. 9 and Table 5, 5 days).

To determine whether untreated scaffolds could support eventualresurfacing by RPE, assays were carried out to examine cell behavior onuntreated scaffolds that were cultured in RPE complete media for 7 days.Cells were seeded at the same density as that onto Bruch's membrane(3164 cells/mm²).

RPE fully resurfaced the untreated scaffold although the cells did notappear to density arrest by this time point (FIG. 10, arrows point toareas of multilayer formation). It was observe similar multilayerformation in RPE seeded onto tissue culture plastic and onto glasscoverslips.

PCL scaffolds can support initial fetal RPE attachment and resurfacingif exposed to sfBCE-CM as a substrate coating the scaffold or as mediaoverlying seeded cells. Although untreated scaffolds may supportlong-term survival of RPE in serum-containing media, modification of thescaffold or addition of ECM ligands may be necessary to supportdifferentiated cell monolayers.

TABLE 5 Fetal RPE behavior on PCL scaffolds that were either untreatedor soaked in serum-free BCE conditioned media (sfBCE-CM). Time inScaffold Culture Treatment Media Cell Behavior 1 day None DMEM Fewrounded cells None sfBCE-CM Many cells, many are spread sfBCE-CM DMEMMany cells, many are spread BCE-ECM on DMEM Many cells, majority plasticare spread 5 days None 2 d DMEM, 3 d RPE Few rounded cells completemedia None 2 d sfBCE-CM, 3 d Fully resurfaced RPE complete mediasfBCE-CM 2 d DMEM, 3 d RPE Fully resurfaced complete media 7 days NoneRPE complete Fully resurfaced, some media multi-layering BCE-ECM on RPEcomplete Fully resurfaced, plastic media monolayer For 1-day studies,cells on untreated scaffolds were cultured in sfBCE-CM or DMEM; cells onsfBCE-CM-soaked scaffolds were cultured in DMEM. For 5 day studies,cells were cultured on untreated or sfBCE-CM-soaked scaffolds for 2 daysin DMEM or sfBCE-CM followed by media change to RPE complete media. For7-day studies, untreated scaffolds were cultured in RPE complete media.Cell behavior on BCE-ECM-coated culture dishes (no scaffold controls) isincluded for day-1 and day-7 data for comparison.

Example 12 Identification of Bioactive Fractions that Support Cells onHuman Aged and AMD Bruch's Membrane and Molecules of BCEC-CM

In this example, BCEC-CM was fractionated to identify fractions havingtherapeutic activity. Briefly, BCEC-CM was collected from passage-2 BCECafter 72 hour exposure to Madin-Darby Bovine Kidney Maintenance Medium.The collected BCEC-CM was subject to ultrafiltration utilizingcentrifugal filters of sizes ranging from 3 to 300 kDa. Afterseparation, the fractions were tested for bioactivity by analyzing RPEsurvival on human submacular Bruch's membrane explants established fromaged and AMD donor eyes. The protein component of the bioactive fractionwas analyzed by mass spectrometry.

The bioactive fraction was identified as those molecules found in thefiltrate generated after ultrafiltration using a 50 kDa filter. Massspectrometry of the 50 kDa filtrate of two different BCEC-CMpreparations identified 72 common secreted proteins, including 5 growthfactors. Subfractionation of the 50 kDa filtrate showed decreasedbioactivity in the filtrate after ultrafiltration utilizing a 30 kDafilter, indicating some bioactivity was contained in the 30-50 kDafraction, and complete loss of bioactivity after removal of the 10-50kDa fraction. Ultrafiltration of the 50 kDa fraction utilizing a 3 kDafilter also showed complete loss of activity in the retentate (3 kDa-50kDa), indicating that bioactivity was present in the <3 kDa filtrate.

Bioactive molecules were found in a fraction generated by molecularweight cut off filtration. This bioactive fraction is comprised ofmolecules found in the fraction generated after filtration using a 50kDa filter. Bioactive molecules supporting long-term survival of cellson aged and AMD submacular Bruch's membrane are found in severalsubfractions of the 50 kDa fraction: a low molecular weight subfraction(below 3 kDa) and a 10-50 kDa subfraction. This finding indicates thereare at least two bioactive molecules in BCEC-CM. Subsequent massspectrometry analysis of the protein component of the 50 kDa fractionidentified four candidate growth factors (proteins that stimulate cellsin a variety of ways including growth stimulation, cell deathprevention, and cell functionality and maturity acquisition). Moleculesfound in the low molecular weight fraction support rapid cellattachment, spreading, and growth in cell culture.

Preliminary two-dimensional gel and mass spectrometry spot ID analysis(see FIG. 11, Table 6 below) showed abundant large molecular weightextracellular matrix ligands (collagens and fibronectin). Thesemolecules (specifically, fibronectin) have been shown to support RPEattachment in cell culture and to support initial attachment on Bruch'smembrane. To determine if high molecular weight components contribute tocell survival on Bruch's membrane, RPE survival was analyzed in largemolecular weight retentates. Molecular cut removal of low molecularweight components showed little or no bioactivity in the retentatefractions (see FIG. 12). The results of testing the high molecularweight retentates (FIG. 12) indicate that a low molecular weightfraction in the 3 kDa filter filtrate must be present in order forBCEC-CM to show complete bioactivity.

TABLE 6 Proteins Obtained by Molecular Cut Filtration and Identified byMass Spectrometry IPI ID Protein MW Gene Function IPI00691126 C-X-Cmotif chemokine 6 12 CXCL5 cytokine IPI00699064 DKK3 protein 38 DKK3cytokine IPI00714868 Protein FAM3C 25 FAM3C cytokine IPI00839037Uncharacterized protein 13 PF4 cytokine IPI00696930 Uncharacterizedprotein 55 EFEMP1 enzyme IPI00702154 Lysyl oxidase-like 1 65 LOXL1enzyme IPI00710136 Angiogenin-1 17 ANG enzyme IPI00760446 Ribonuclease,RNase A family, 4 17 RNASE4 enzyme IPI00686503 Platelet-derived growthfactor subunit 24 PDGFA growth factor IPI00698668 Connective tissuegrowth factor 38 CTGF growth factor IPI00706240 growth arrest-specific 674 GAS6 growth factor IPI00731393 PDGFD protein 42 PDGFD growth factorIPI01018572 Insulin-like growth factor I variant 2 21 IGF-1 growthfactor IPI00714018 Insulin-like growth factor I 17 growth factorIPI00685095 Cystatin-C 16 CST3 other IPI00685504 Alpha 1 type VIIIcollagen (Fragment) 73 COL8A1 other IPI00688802 Uncharacterized protein136 NID1 other IPI00688875 Follistatin-related protein 3 28 FSTL3 otherIPI00690094 Galectin-1 15 LGALS1 other IPI00692839 Uncharacterizedprotein 44 other IPI00698975 SPARC 35 SPARC other IPI00702294Plasminogen activator inhibitor 1 45 other IPI00704150 VitaminK-dependent protein S 75 PROS1 other IPI00705697 Insulin-like growthfactor-binding 34 IGFBP2 other IPI00706624 Procollagen C-endopeptidase48 PCOLCE other IPI00707101 Alpha-2-HS-glycoprotein 38 AHSG otherIPI00707467 Follistatin-related protein 1 35 FSTL1 other IPI00707932collagen alpha-2(VIII) chain 67 COL8A2 other IPI00708244 Collagenalpha-2(I) chain 129 COL1A2 other IPI00708990 Uncharacterized protein148 LTBP1 other IPI00709059 Angiopoietin-related protein 7 39 ANGPTL7other IPI00709084 Metalloproteinase inhibitor 1 23 TIMP1 otherIPI00710025 Factor XIIa inhibitor 52 other IPI00710385 Prolargin 44PRELP other IPI00710453 Matrix Gla protein 12 MGP other IPI00711862Epididymal secretory protein E1 17 NPC2 other IPI00712084Thrombospondin-1 130 THBS1 other IPI00712366 Fibromodulin 43 FMOD otherIPI00712524 collagen, type IV, alpha 2, partial 165 COL4A2 otherIPI00713428 ADM 21 ADM other IPI00713573 Uncharacterized protein 109COL6A1 other IPI00716121 Pigment epithelium-derived factor 46 otherIPI00718311 Isoform 1 of Proactivator polypeptide 58 PSAP otherIPI00718620 Insulin-like growth factor-binding 28 IGFBP4 otherIPI00730859 Uncharacterized protein 139 LTBP3 other IPI00731756Uncharacterized protein 24 SCG5 other IPI00824031 LTBP1 protein 147LTBP1 other IPI00838716 Uncharacterized protein (Fragment) 102 CHRDother IPI00840999 PCSK1N protein 27 PCSK1N other IPI00883474 gelsolin a86 GSN other IPI00905045 collagen alpha-1(XI) chain 182 COL11A1 otherIPI00906401 Uncharacterized protein (Fragment) 25 IGFBP6 otherIPI00733988 collagen type 5 alpha 1-like 30 other ECM IPI00716123Mimecan 34 OGN other ECM IPI00685447 72 type IV collagenase 74 MMP2peptidase IPI00705266 Uncharacterized protein 102 ADAMTS5 peptidaseIPI00712538 HtrA serine peptidase 1 67 HTRA1 peptidase IPI00713459 Adisintegrin and metalloproteinase 136 ADAMTS3 peptidase IPI00713505Complement C3 (Fragment) 187 C3 peptidase IPI00714873 Serine protease 2342 PRSS23 peptidase IPI01004181 bone morphogenetic protein 1-like 107BMP1 peptidase IPI00717574 Carboxypeptidase E 53 peptidase IPI00689362Transthyretin 16 TTR transporter IPI00690534 Serotransferrin 78 TFtransporter IPI00708398 Uncharacterized protein 70 ALB transporterIPI00712693 Apolipoprotein E 36 APOE transporter IPI00713780Uncharacterized protein 24 APOD transporter IPI00715548 ApolipoproteinA-I 30 APOA1 transporter IPI00866855 IGFBP7 protein 29 IGFBP7transporter IPI00697184 retinol-binding protein 4 23 Secretedtransporter IPI00867435 NID2 protein 143 Secreted? other

Testing of the filtrates revealed complete bioactivity can be retainedin filtrates utilizing the 50 kDa filter, indicating that thebioactivity is comprised of molecules of molecular weight at or near 50kDa and lower. Although there appears to be a trend towards decreasednuclear density in the <30 kDa filtrate, the difference is notsignificant (Kruskal-Wallis One Way Analysis of Variance on Ranks,P=0.092).

The results of these examples indicate that there are a minimum of twobioactive molecules in BCEC-CM, one found in the <3 kDa filtrate and onefound in the 10-50 kDa fraction and that bioactive molecules in bothfractions must be present to ensure cell survival on Bruch's membrane.

Example 13 Differentiation Medium for RPE

In this example, studies were performed to determine whether fetal RPEmature more rapidly in BCEC-CM compared to standard RPE medium, whereonset of maturity was based on mRNA expression of late RPEdifferentiation markers, bestrophin and RPE65. More specifically, fetalRPE were seeded at a high seeding density (3164 cells/mm²) onto BCEC-ECMcoated tissue culture dishes and maintained in culture for 21 days inthe aforementioned RPE medium or BCEC-CM. The media were changed3×/week. At day-21, the cells were harvested off the tissue culturedishes for real time PCR mRNA analysis of bestrophin and RPE65.

It was found that the cells cultured in BCEC-CM appeared to have patchesof cells that looked more mature (morphologically) than cells in the RPEmedium. In contrast, the cells in the RPE medium appeared to be moreuniform in appearance. The cells cultured in BCEC-CM expressedapproximately 10× more RPE65 and 50× more bestrophin mRNA than the cellscultured in the RPE medium.

Example 14 Bovine Corneal Endothelial Cell Conditioned Medium as StorageMedium

In this example, assays were carried out to examine the properties of CMin preserving confluent fetal RPE monolayers under conditions likely tooccur during shipping and storage of cells attached to a substrate priorto use in patients.

a. Cell Behavior after Storage in RPE Medium, Optisol, and CM (Batch34AB)

In this assay, the substrate was tissue culture plastic (TCP) or TCPcoated with bovine corneal endothelial cell extracellular matrix (ECM).Optisol is a medium developed for corneal storage. Passage 2 fetal RPEwere seeded on ECM or directly on TCP and cultured in standard RPEmedium for 11 days. At the start of the storage period, cells wereplaced in one of the following three media:

(i) RPE medium: DMEM base medium (HEPES, inorganic salts, amino acids,vitamins, glucose, sodium pyruvate), bFGF, fetal bovine serum,glutamine, gentamicin, and fungizone;

(ii) Optisol (Bausch and Lomb, Inc., proprietary cornea preservationmedium) “Optisol base powder”, chondroitin sulfate, dextran, sodiumbicarbonate, antibiotics and fungizone, sodium pyruvate, glutamine,mercaptoethanol, and amino acids; and

(iii) CM: MDBK-MM base medium (Sigma Aldrich proprietary formulationincluding HEPES, human recombinant peptides (insulin and possiblyothers), amino acids, and sodium bicarbonate), CM harvested after 3 dayexposure of MDBK-MM to confluent bovine corneal endothelial cellcultures.

Experiments were carried out at 4° C. (refrigerator) and at roomtemperature in sealed culture plates or dishes with no media change.Although it is unlikely a surgical site will have a 37° C. CO₂ incubatorsuitable for storing cells for patient use, the viability of storingfetal RPE was compared in such an incubator. In these preliminarystudies, the onset of cell death was estimated by initial appearance ofdefects in the RPE monolayer.

TABLE 7 Onset of cell death after storage in Optisol, CM, or RPE mediumSubstrate/ temperature Optisol CM RPE TCP/4° C. <7 days ~21 days ~21days TCP/RT <3 days >21 days <7 days ECM/4° C. <10 days <10 days <10days ECM/RT <3 days <3 days ECM/37° C. <7 days <7 days (unsealed)TCP/37° C. 14-<21 days 14-21 days (unsealed)

The results were shown in Table 7. It was found that Optisol wasrelatively poor at preserving RPE viability. All cells stored in Optisolwere of abnormal morphology at early time points. Both CM and RPE mediagenerally maintained fetal RPE viability to a similar degree except CMwas better at preserving RPE at room temperature if the cells were ontissue culture plastic. Fetal RPE cultured on an extracellular matrixthat supports rapid cell attachment and growth in cell culture, showedpoor viability regardless of the storage medium.

b. Storage of Confluent RPE Cultures in CM (Batch 58) Molecular CutFractions

In this assay, confluent fetal RPE cultures (passage 3 or 4, 7-13 daysin culture) grown on tissue culture plastic were stored at roomtemperature or 4° C. in sealed 48 well plates with no medium change forup to 7 days. The storage media were RPE medium and CM, including 3 kDand 50 kD CM molecular cut filtrates. Live/death assessment wasperformed at day-3 or day-7. The results are shown in Table 8 below,where the values reflect the days in storage when loss of cells in theRPE monolayer was observed.

TABLE 8 Onset of cell death after storage in RPE medium and CM molecularcut fractions Storage medium 4° C. RT RPE 3-4 D 3-4 D  3K CM 4 D-<7 D >7D 50K CM 4 D-<7 D 7 D->7 D Uncut CM 4 D-<7 D >7 D

It was found that onset of cell death was sooner than in the previousstudy (Table 7 vs. Table 8), possibly due to variability between CMbatches and/or the fetal RPE passage number and time in culture. Anadditional consideration for comparisons between experiments for storageat room temperature is the variability in ambient temperature. RPEmedium effective storage times were consistently short for bothtemperatures. Storage times in CM, including CM fractions were longerthan RPE for storage at room temperature. The 3 kD and 50 kD filtratesstorage times were similar to CM that had not be subject to molecularcut filtration (“uncut CM”).

Example 15 Storage of Cell Suspensions

In this example, assays were performed to examine the ability of CM forshipping and storing fresh cell suspensions on wet ice or ambienttemperature. Such ability offers an alternative to using frozen cellsfor patient transplants. Frozen cells require storage in liquidnitrogen, shipment on dry ice or under liquid nitrogen, and thaw, rinse,and resuspension in delivery vehicle for patient use. Cells recoveredfrom thaw attach and grow slower than fresh cells with some cell deathoccurring from freezing and subsequent manipulations. Ideally, freshcells would be stored in a solution similar to that of the deliveryvehicle so no or few manipulations are required.

a. Cell Viability after Storage in CM vs. RPE Medium

Previous studies indicated that RPE undergo apoptosis if not attached toa suitable substrate by 24 hours (Tezel et al. Graefes Arch Clin ExpOphthalmol 1997; 235:41-47). In this assay, studies were performed todetermine if cell suspensions can retain any degree of viability afterstorage.

Briefly, fetal RPE cell suspensions (100,000 in 100 ul) in the CM or RPEmedium (contains fetal bovine serum, glutamine, and basic fibroblastgrowth factor) were placed in sealed microfuge tubes. The tubes werestored at room temperature or 4° C. for 1, 2, 3, or 7 days. At the endof the storage period, the numbers of live and dead cells weredetermined by trypan blue staining and the remaining cells plated ontissue culture plates and cultured in fresh storage medium to assessviability.

FIG. 16 illustrates cell viability, expressed as the percent of livecells at the end of each storage period, for fetal RPE suspensions in CMor RPE medium at room temperature (RT) or 4° C. The results showed thatfetal RPE suspensions can retain a high degree of viability when storedin sealed microfuge tubes at 4° C. and room temperature for up to 3 daysin storage. At 7 days in storage, cells stored in the RPE medium at 4°C. showed a marked drop in percent of live cells. At room temperature,cells were clumped in both media, making viability assessment difficult.Harvested cells from each storage time showed rapid attachment,spreading, and growth when cultured on tissue culture plastic. Allcultures were confluent by day-7 in culture except for the 7-daystorage, room temperature cells that were stored in CM. These data showthat it is possible to store cell suspensions for a short period of timewith little loss in cell viability.

b. Storage of Cell Suspensions in Molecular Cut Fractions of CM (CMBatch 58)

The studies of part a in Example 14 above were repeated with additionalstorage in 3 kD and 50 kD CM molecular cut filtrates. To determine thechange in cell numbers with time in culture, time 0 viability countswere performed for each tube of cells to be stored. To better assesscell numbers in tubes stored at room temperature, cell clumps weretreated with trypsin for the 3 and 7 day storage time points. Cellviabilities in all media were below the levels measured at time 0 atdays 3 and 7.

It was found that, consistent with the previous experiment (FIG. 16),poorest viability was observed in cells stored at 4° C. in the RPEmedium (FIG. 17). At room temperature storage, all cells maintainedviability levels above that measured at time zero (FIG. 18). Cellsstored in CM and CM fractions showed better preservation of live cellsthan those stored in RPE medium at 4° C. (FIG. 19). The change in cellnumbers in the RPE medium at day-7 is consistent with the drop in cellviability at this time point. The day-1 data are not shown since thecounting method was not the same as days 3 and 7. The increase in livecell numbers at day-3 compared to time 0 indicated that the cells weredividing. Cells in 50 kD CM appear to maintain cell division at day-7.

As shown in FIG. 20, storage in 3 kD CM and the RPE medium at roomtemperature showed increased number of live cells at day-1 while livecell numbers dropped to similar levels in 50 kD CM and uncut CM. Similarto storage at 4° C., cells in the RPE medium show a drop in live cellswith time in culture. The cells in 50 kD CM appeared to be dividingduring 3 and 7 day storage periods with number of live cells at day-7well above the time 0 levels. The cells in uncut CM showed a marked risein the number of live cells at day-7 compared to time 0 levels.

Example 16 CM as Culture Medium

In this example, assays were performed to examine whether CM could offeran advantage over a standard RPE culture medium as a defined, serum-freemedium.

a. Comparison of Cell Behavior in CM vs. RPE Medium

Studies of fetal RPE behavior (when cultured in CM vs. RPE medium) showthat CM can support fetal RPE to some degree when cultured directly ontissue culture plastic. It was found that cells in CM rapidly attached,spread, and grew to confluence. The ability of CM to support cells inlong-term cultures is highly variable and may depend on the batch of CMand/or the fetal RPE starting culture (passage number, length of time inculture prior to harvest). In some CM cultures, some cell death occurredbetween day-14 and day-21. In other cases, CM cultures were similar insize at 14 and 21 days although the cells may be more pigmented atday-21.

Lastly, it was found that some CM supported cells to a higher degreewith mature cells at day-21 compared to day-14. In RPE medium, some celldeath and/or cessation of cell division appeared to occur between day-7and day-21 in some cultures as the cultures were similar in appearanceor the cells are larger at day-21. Additionally, the presence ofpigmented clumps of dead cells could be seen in many cultures at thesetime points. In comparing parallel cultures of CM and RPE medium, CMappeared to preserve cell viability longer than RPE medium. Studies arein progress to determine cell behavior in CM vs. RPE medium on singlehuman ECM proteins (e.g. laminin, collagen I, collagen IV).

The set of figures in FIGS. 21A-F are parallel cultures at differentculture times. It was found that fetal RPE attached and spread rapidlyin early cultures. As shown in the figures, by 7 days, both culturesappeared similar. At day-14, the appearance of mature RPE could be seenin both cultures; some of the cells were very small and appeared to haverounded (columnar) vs. flat surfaces. There appeared to be more of thesehighly differentiated cells in the CM culture vs. RPE medium culture.The white arrow points to a cluster of highly pigmented RPE. These couldbe dead/dying RPE that are shed from the culture. By 21 days, the cellswere larger in both media, indicating that some cell death occurred.

b. Culture with Limited Viability of RPE in RPE Medium

In this example, additional assays were performed to examine effects ofCM and the RPE medium on cell behavior.

In a set of parallel cultures (see FIG. 22), RPE in the RPE medium ontissue culture plastic showed some degree of cell death as early as 8days in culture with the appearance of pigmented clusters on top of theRPE monolayer. The cells in the RPE medium were of similar size at allthree time points. The RPE appeared to be smaller when cultured in CMindicating cell division occurred.

c. Expression of RPE Differentiation Markers in Cells Cultured in CM vs.RPE Medium

Assays were carried out to examine mRNA expression levels 21 days afterseeding on tissue culture plastic utilizing a different CM batch thanthat used in Example 13. It was found that the fetal RPE cultured in CMexpressed 9.6× more Bestrophin and 1.28× more RPE65 than the cellscultured in the RPE medium. Western blot also showed Bestrophin proteinpresent as strong bands in 14- and 21-day CM cultures with faint (ifpresent at all) bands in parallel RPE medium cultures at the same timepoints.

d. Culture of RPE in 3 kD Filtrate

While CM low molecular weight components alone do not support cells onBruch's membrane, the 3 kDa filtrate alone supported attachment andspreading of human fetal RPE on human collagen I, a major component ofthe inner collagenous layer of Bruch's membrane, as well as on uncoatedtissue culture plastic although not to the same degree.

On collagen I, cell spreading was observed initially by day-1 and byday-3 in culture, ≧50% of RPE are spread. Cells reached confluence by 3days or later, depending on the CM batch. On tissue culture plastic, RPEattachment, spreading, and growth occurred but, depending on CM batch,to a lesser degree than was observed on collagen I, with the onset ofspreading later on tissue culture plastic. The 10-50 kDa fraction alonesupported only limited attachment and spreading on both tissue cultureplastic and collagen I at early times in culture with only a fewelongate cells observed at day-3. Addition of the 3 kDa filtrate to thisfraction restored the activity to the level observed in <50 kDafiltrate. These studies indicate that the 3 kDa filtrate containsbioactive molecules that are necessary for cell attachment and growth inlong-term cell culture.

Example 17 Enhancing Cell Survival on Bruch's Membrane in Eyes Affectedby Age and AMD

In this example, assays were carried out to determine whether BCEC-CMcan support transplanted cells on aged and AMD Bruch's membrane (BM).

Currently, no proved treatment options exist for patients withgeographic atrophy, an advanced form of AMD. For selected patients withextensive drusen or geographic atrophy threatening the fovea, celltransplants might prevent central vision loss through replacement ofdysfunctional or dead RPE cells. Anti-vascular endothelial growth factortherapy is currently the best treatment available for AMD-associatedCNVs, but randomized studies indicate that only 25-40% of treatedpatients experience at least moderate visual improvement. Thus, eventoday, a significant number of patients become blind despite theavailability of pathway-based therapy for AMD-associated CNVs. If celltransplants could prevent CNV development or rescue photoreceptorsfollowing CNV excision, then these transplants also might have an impacton CNV-related blindness.

A major obstacle to the success of RPE transplants in AMD patients isthe failure of transplanted RPE cells to survive and become functionalin the diseased AMD eye. RPE transplantation in patients with AMD(atrophic and neovascular) typically has produced limited visualrecovery regardless of the type of cell transplanted (e.g., autologousor allogeneic, adult or fetal RPE) or whether the cells are transplantedwith or without choroid. In contrast, RPE transplantation in animalmodels of retinal degeneration has been proved to rescue photoreceptorsand preserve visual acuity. Although animal studies validate celltransplantation as a means of achieving photoreceptor rescue, animportant distinction between humans with AMD and laboratory animals inwhich RPE transplantation has been successful is the age- andAMD-related modifications of the surface on which human RPE reside insitu (i.e., Bruch's membrane), which may have a significant effect onRPE graft survival. Evidence from human donor eye organ cultureexperiments indicates that healthy RPE cannot survive for an extendedperiod of time on aged submacular Bruch's membrane, and the poorestsurvival is observed on AMD Bruch's membrane. These in vitro studieswere performed on human submacular Bruch's membrane with no treatment toimprove cell survival. Previous studies to improve cell survival on agedBruch's membrane included adding ECM ligands singly or in combination to“coat” Bruch's membrane, detergent treatment to eliminate debrisaccumulated within Bruch's membrane followed by ECM ligand coating, andresurfacing Bruch's membrane with a cell-deposited matrix. The first twomethods showed limited improvement in attachment and early survival.Long-term survival was not demonstrated. The last method improvedlong-term cell survival more than 200%. However, from a therapeuticstandpoint, resurfacing submacular Bruch's membrane with thecell-deposited ECM was problematic due to the inability to solubilizeECM components in a manner compatible with clinical application. Thesestudies demonstrate the need for development of a method to improvelong-term cell transplant survival in AMD patients.

BCECs secrete an ECM that supports rapid attachment, growth, anddifferentiation of RPE. During BCEC-ECM formation, in addition to basalsecretion, BCECs secrete ECM components into the overlying medium,including collagens, proteoglycans, and entactin/nidogen. Secretion ofECM components into the overlying medium is most abundant in earlypassage cells and exceeds basal ECM deposition in quantity. Sincesoluble ECM can affect cell shape and metabolism in addition tostimulating production of ECM molecules, the presence of these proteinssuggests that conditioned medium harvested from BCEC cultures could be asource of cell-supporting soluble proteins and, if effective, could leadto development of an adjunct to cell-based therapy for AMD. In thisexample, assays were performed to characterize the behavior of RPE cellstransplanted onto Bruch's membrane of aged and AMD donor eyes culturedin BCEC-CM or CM vehicle utilizing a previously characterized humansubmacular Bruch's membrane bioassay.

Material and Methods

Conditioned Medium Preparation

Cow eyes (ages 6 months -3 years) were obtained from localslaughterhouses. Each globe was rinsed briefly in 70% ethanol, and thecornea was separated from the rest of the globe by making acircumferential cut anterior to the limbus. The cornea was rinsedquickly in PBS and positioned with the epithelial surface down on asterile support placed on a Petri dish. The cup formed by the cornea wasfilled with 0.05% trypsin-0.02% EDTA (Invitrogen-Gibco, LifeTechnologies, Carlsbad, Calif.) and placed in a 37° C., 10% CO2incubator for 30-60 minutes. BCECs were scraped off gently using a bluntmetal spatula and collected into a 15 ml tube containing Dulbecco'smodified Eagle's medium (DMEM, Cellgro, Manassas, Va.) supplemented with2 mM glutamine, 15% fetal bovine serum (FBS), 2.5 μg/ml amphotericin B,50 μg/ml gentamicin, and 1 ng/ml bFGF (all from Invitrogen-Gibco)(termed “RPE medium”). Cells were spun down, resuspended in RPE medium,seeded onto 60 mm dishes, and cultured at 37° C. in 10% CO2. Cultureswere passaged at confluence. For BCEC-CM harvest, passage-2 or -4 cellswere cultured in RPE medium with 10% FBS and 5% donor bovine serum(Invitrogen-Gibco), instead of 15% FBS, until confluent. BCEC-CM wasobtained by incubating confluent BCEC cultures for 72 hours inMadin-Darby Bovine Kidney Maintenance Medium (MDBK-MM, Sigma-Aldrich,St. Louis, Mo.) supplemented with 2.5 μg/ml amphotericin B and 50 μg/mlgentamicin. The vehicle, MDBK-MM (hereafter referred to as “CMvehicle”), is a serum- and protein-free, defined medium designed formaintaining high-density cultures of MDBK cells. Following collection,BCEC-CM was centrifuged briefly to remove cellular debris, and thesupernatant was stored at −80° C. Twelve batches of BCEC-CM were used inthis study.

Cell Culture

RPE were isolated from fetal eyes (Advanced Bioscience Resources, Inc.,Alameda, Calif.; gestational age 18-22 weeks) or adult eyes (donor age58, 71, 78 yrs.) after incubation of RPE/choroid pieces in 0.8 mg/ml(fetal eyes) or 0.4 mg/ml collagenase type IV (Sigma-Aldrich) (adulteyes) as described previously. RPE were cultured in RPE medium on bovinecorneal endothelial cell-extracellular matrix (BCEC-ECM)-coated tissueculture dishes prepared in this laboratory according to a previouslydescribed protocol. After achieving confluence, primary fetal RPEcultures were passaged at a 1:6 split ratio onto BCEC-ECM-coated dishesusing 0.25% trypsin-EDTA to harvest the cells. Subsequent cultures werepassaged at a 1:4 split ratio. Adult RPE seeded onto Bruch's membranewere from day-11-15 primary cultures; fetal RPE were harvested fromcultures of passage 1-3, 3-7 days in culture after seeding. Humanembryonic stem cell-derived RPE (hES-RPE, Advanced Cell Technology,Worcester, Mass.), were established from a stem cell culture designatedas MA09. Cells were maintained in MDBK-MM medium (Sigma-Aldrich) untilremoval from flasks and seeding onto Bruch's membrane explants. Cells ofpassage-32 as stem cells and passage-2 or -3 as hES-RPE were utilizedand were removed from culture dishes using trypsin/EDTA after 50-85 daysin culture.

Bruch's Membrane Organ Culture

Adult donor eyes were received from the Lions Eye Institute forTransplant and Research (Tampa, Fla.) and eyebanks placing donor eyesthrough their website (Ocular Research Biologics System (ORBS),orbsproject.org), Midwest Eyebanks (includes eyebanks in Illinois,Michigan, and New Jersey), the San Diego Eyebank (San Diego, Calif.),and eyebanks placing tissue through the National Disease ResearchInterchange (NDR1, Philadelphia, Pa.). Acceptance criteria for donoreyes included: 1) death to enucleation time no more than 7 hours; 2)death to receipt time no more than 48 hours; 3) no ventilator supportprior to death; 4) no chemotherapy within the last 6 months prior todeath; 5) no radiation to the head within the last 6 months prior todeath; 6) no recent head trauma; 7) no ocular history affecting theposterior segment except for AMD. These acceptance criteria have beenfound in previous studies to yield well-preserved explants. Posteriorsegments were examined through a dissecting microscope for submacularpathology and documented by photography. A previously published methodwas used to create inner collagenous layer (ICL) surfaces by mechanicaldebridement. Six-millimeter diameter corneal trephines (Bausch and Lomb,Rochester, N.Y.) were used to create macula-centered, Bruch's membraneexplants. Explants were placed in wells of 96-well plates for cellseeding and organ culture. Cells were seeded at a seeding density of3164 cells/mm², a seeding density that has been shown to yield amonolayer of cells on a 6 mm diameter Bruch's membrane explant in organculture one day after seeding. Explants were harvested at day-21, fixedin phosphate-buffered 2% paraformaldehyde and 2.5% glutaraldehyde,bisected, and processed for light or scanning electron microscopy.

Scanning Electron Microscopy (SEM)

Explant halves for SEM were post-fixed in phosphate buffered osmiumtetroxide, dehydrated using a graded series of ethanol, critical pointdried (Tousimis, Rockville, Md.), and sputter-coated (Denton,Moorestown, N.J.) according to standard SEM protocols. SEM imageacquisition (JEOL JSM 6510, Tokyo, Japan) was performed with routinephotography at 30×, 50×, 200×, and 1000×. SEM evaluation of Bruch'smembrane involved assessment of cell surface morphology and, in areasnot resurfaced by cells, the level of Bruch's membrane exposed bydebridement.

Light Microscopy (LM)

Bruch's membrane explant halves processed for histology were embedded inLR White (Electron Microscopy Sciences, Hatfield, Pa.); 4-6 sections of2 μm thickness were mounted on slides, dried overnight, and stained with0.03% toluidine blue (Electron Microscopy Supply). LM evaluation focusedon RPE morphology (cell shape, density, pigmentation, polarization) andevaluation of Bruch's membrane and choroid. Nuclear density counts wereperformed to assess treatment success quantitatively, comparing pairedexplants from fellow eyes. Nuclear density counts were performed bycounting the number of RPE nuclei in intact cells in contact withBruch's membrane in the central 3 mm of 4-5 non-consecutive slides(approximately every 5th slide). Linear measurements of Bruch's membranein the analyzed area were obtained by digital image acquisition andmeasurement with the freehand line tool using NIH Image J(http://rsb.info.nih.gov/ij/index.html). Nuclear density was expressedas the number of nuclei per mm of Bruch's membrane.

Statistical Analysis

Statistical differences between pairs were determined by Wilcoxon SignedRank tests. For comparisons between time points and comparison betweengroups, existence of significant differences was determined byKruskal-Wallis One Way Analysis of Variance on Ranks. If significancewas observed, All Pairwise Multiple Comparison Procedures testing(Dunn's method) determined the significance between pairs of groups.Comparisons between two groups in unpaired studies were by Mann-WhitneyRank Sum tests. Comparisons between ages of two groups were by unpairedt-tests or between multiple groups by One Way ANOVA. Ages are indicatedas mean age with standard deviation. A P value <0.05 was consideredstatistically significant.

Extracellular Matrix Deposition

Fetal RPE (3164 cells/mm²) were seeded onto tissue culture-treatedplastic (48-well plates) or Bruch's membrane and cultured in BCEC-CM orRPE medium. ECM on tissue culture plates was analyzed at day-7, -14, and-21 (N=3). ECM on Bruch's membrane (6 donor pairs; three pairs withextensive drusen; 3 pairs normal; mean donor age, 79.2±3.17 yrs.) wasanalyzed at day-21 only. Primary antibodies were: mouse monoclonalcollagen IV (1:500 dilution, Sigma-Aldrich), rabbit polyclonal laminin(1:25 dilution, Sigma-Aldrich), and mouse monoclonal fibronectin (1:50dilution, Abcam, Cambridge, Mass.). Secondary antibodies werefluorescein (FITC)-conjugated goat anti-mouse IgG (H+L) and rhodamine(TRITC)-conjugated goat anti-rabbit IgG (H+L) applied at 1:50 dilution(both from Jackson ImmunoResearch Laboratories, West Grove, Pa.). Allantibodies were diluted in 2% normal goat serum, 0.3% Triton X-100 (bothfrom Sigma-Aldrich) in phosphate buffered saline (PBS).

Cell culture: Cells were removed from culture wells by incubating in0.02M NH4OH for 5 minutes followed by rinsing in PBS. The exposed ECMwas fixed for 15 minutes in cold 4% paraformaldehyde followed by 3 PBSwashes. Wells were then incubated for 45 minutes at room temperature inblocking solution (2% normal goat serum, 0.5% BSA in PBS). Primaryantibodies were applied to culture wells and incubated for 2 hours atroom temperature. After washing with PBS plus 0.3% triton, secondaryantibodies were applied, and wells were incubated for 1 hour at roomtemperature. After washing with PBS plus triton, mounting medium(Vectashield, Vector Laboratories, Burlingame, Calif.) was added to thewells. Epifluorescence images for each protein at the same time pointwere photographed at the same exposure to determine relative differencesin the amount of deposited protein using an inverted microscope equippedwith the appropriate fluorescein and rhodamine filters (10× neoflaurobjectives, Axiovert, Carl Zeiss, Thornwood, N.Y.). Followingimmunostaining photography, ECM was stained with 0.1% Ponceau S(Sigma-Aldrich) for 10 minutes at room temperature and photographedusing a 32× phase objective.

Bruch's membrane: At day-21, live RPE were imaged by calcein staining(Live/Dead viability/toxicity assay, Molecular Probes, Eugene, Oreg.) todetermine surface coverage by cells. Following a brief rinse in PBS,explants were incubated in 2 μM calcein for 1 hour at room temperature.Explants were rinsed briefly, then photographed at 2.5× magnificationsusing a fluorescence microscope equipped with a fluorescein filter set(Axiophot, Carl Zeiss). Montages of calcein-imaged explants were createdin Photoshop CS4 (Adobe Systems, Mountain View, Calif.). After calceinimaging and RPE removal by 5 minute incubation in 0.02M NH4OH, explantswere fixed in 4% paraformaldehyde for 1 hour at 4° C. Following washingin PBS, explants were blocked at room temperature for 45 minutes. Theexplants were then bisected prior to immunostaining, and the surface ofBruch's membrane was immunostained for laminin and collagen IV (onehalf) and fibronectin and laminin (other half) or cut into thirds withthe third piece used for controls. Primary antibodies were applied toexplants, which were then incubated overnight at 4° C. The followingday, explants were rinsed with PBS, and secondary antibodies wereapplied. Explants were incubated for 2 hours at room temperaturefollowing by washing with PBS. Explants were stored and examined inmounting medium. Single images or z-stacks were acquired from thesurface of Bruch's membrane using a 40× water immersion lens on aconfocal microscope (LSM510, Carl Zeiss, Thornwood, N.Y.). Lasers linesand corresponding emission filters were: 488 nm excitation, 505-530 nmband pass filter for FITC; 543 nm excitation, 560-615 nm band passfilter for rhodamine. Following confocal microscopy evaluation, explantswere processed for SEM.

Results Effect of BCEC-CM on Long-Term Cell Survival on Aged and AMDBruch's Membrane

RPE derived from human embryonic stem cells (hES-RPE), fetal RPE, andaged adult RPE were seeded onto the inner collagenous layer ofsubmacular Bruch's membrane of donor eyes age 62 and older (see Table 9below for donor information) and cultured in BCEC-CM or CM vehicle(representative images, FIGS. 23-27). On explants cultured in CMvehicle, limited resurfacing was seen at day-21 with no or few survivingcells on the majority of explants (hES-RPE, 3 of 6 total explantsresurfaced with few cells, and no cells on the remaining 3 explants;fetal RPE, 20 explants with no or few cells of 22 total explants; adultRPE, 5 explants with no or few single cells of 7 total explants) (FIGS.23-27, A-C). When present, intact cells were large and flat, regardlessof cell type (FIGS. 24C, 27B). Cytoplasmic vacuoles were common. Allexplants cultured in BCEC-CM showed cells remaining at day-21 with manyexplants almost fully (i.e., more than 75% of the surface covered bycells) or fully resurfaced (hES-RPE, 3 explants completely or almostfully resurfaced of 6 total explants; fetal RPE, 16 explants completelyor almost fully resurfaced of 22 total explants; adult RPE, 6 explantsalmost fully resurfaced of 7 total explants). hES-RPE (FIGS. 23 E-G)cells were predominantly monolayered with highly variable morphology andwere larger and flatter than fetal RPE (FIGS. 24-26, E-G). Fetal RPEshowed focal areas of bi- and multilayers with the most extensivemultilayering found in explants that underwent CNV removal prior to cellseeding (FIG. 25E). Resurfacing was extensive and many cells werecompact with abundant expression of well-developed surface apicalprocesses regardless of submacular pathology (FIGS. 22-26E, insets).Adult RPE, generally larger than fetal RPE, were predominantlymonolayered with localized multilayered clumps of cells (FIGS. 27E-G).Adult RPE exhibited abundant short apical processes (FIG. 27E). Sinceadult RPE were from primary cultures, the cells were generally morepigmented than hES-RPE or fetal RPE (FIG. 27G).

The nuclear density of hES-RPE cells grown in BCEC-CM (mean, 20.1 nucleiper mm Bruch's membrane) was significantly higher than that of cellsgrown in CM vehicle (mean, 5.1 nuclei per mm Bruch's membrane)(P=0.031). Fetal RPE nuclear density after culture in BCEC-CM (mean,20.6 nuclei per mm Bruch's membrane) was significantly higher than cellscultured in CM vehicle (mean, 2.2 nuclei per mm Bruch's membrane)(P<0.001). Adult RPE cultured in BCEC-CM nuclear density (mean, 10.0nuclei per mm Bruch's membrane) was also significantly higher than thenuclear density of cells cultured in CM vehicle (mean, 1.2 nuclei per mmBruch's membrane) (P=0.016). (See FIG. 28A.) ANOVA on ranks showedsignificant differences in the median values among the cell typescultured in BCEC-CM (P=0.004). Nuclear densities of fetal RPE andhES-RPE cultured in BCEC-CM were significantly higher than the nucleardensity of adult RPE cultured in BCEC-CM (P<0.05). Fetal RPE and hES-RPEnuclear densities were not significantly different when cultured inBCEC-CM (P>0.05). Nuclear densities of hES-RPE, fetal RPE, and culturedadult RPE were not significantly different when cultured in CM vehicleonly (P=0.060). There were no statistically significant differences inages of donor eye explants between groups (P=0.345: hES-RPE mean donorage, 80.2±8.4 yrs.; fetal RPE mean donor age, 80.2±7.8 yrs.; aged adultRPE mean donor age, 75.7±3.64 yrs.). It was assessed whether RPEsurvival on age-matched AMD vs. non-AMD Bruch's membrane was similar inthe presence of BCEC-CM (FIG. 28B). Explants seeded with fetal RPE onaged Bruch's membrane from eyes without significant AMD changes(including donor eyes with submacular focal RPE hyerplasia and few small(<100 μm) drusen were compared to explants seeded on AMD Bruch'smembrane. AMD donors included donor eyes with CNVs (removed prior tocell seeding with no subsequent debridement), geographic atrophy, and/orextensive large (≧125 μm) drusen. After culture in BCEC-CM, non-AMDdonor eye mean nuclear density was 19.6 nuclei/mm Bruch's membrane, andAMD donor eye mean nuclear density was 21.3. After culture in CMvehicle, non-AMD donor eye mean nuclear density was 2.4 nuclei/mmBruch's membrane, and AMD donor eye mean nuclear density was 2.0. Thedifferences in fetal RPE nuclear density on Bruch's membrane in thepresence of BCEC-CM vs. CM vehicle were statistically significant forboth non-AMD and AMD donors (non-AMD, P=0.004; AMD, P<0.001). For agiven culture medium, the nuclear densities of fetal RPE on non-AMD vs.AMD explants were not significantly different (culture in BCEC-CM,P=0.548; culture in CM vehicle, P=0.231). Ages of the two groups werenot statistically different (P=0.226: aged, non-AMD mean donor age,77.8±5.0 yrs.; AMD mean donor age, 82.2±9.3 yrs.).

RPE Cell Survival Following Different BCEC-CM Culture Times

To determine whether RPE behavior on submacular Bruch's membraneexplants depends on the time of exposure to BCEC-CM, explants seededwith fetal RPE and cultured for differing periods of time in BCEC-CMwere compared to fellow eye explants cultured for the entire incubationperiod (21 days) in BCEC-CM. One explant of the pair was cultured inBCEC-CM for 3-, 7-, or 14-days followed by culturing in CM vehicle tobring the total number of days in culture to 21 (FIG. 29). Fellow eyeexplants were cultured in BCEC-CM for the entire 21-day period. FetalRPE nuclear densities on explants cultured for 3 days in BCEC-CM (meannuclear density, 3.5 nuclei/mm Bruch's membrane) vs. 21 days in BCEC-CM(mean nuclear density, 21.8) were significantly different (P=0.016).Nuclear densities on explants after 7-day BCEC-CM culture (mean nucleardensity, 10.9 nuclei/mm Bruch's membrane) vs. 21 days (mean nucleardensity, 28.0) were significantly different (P=0.008). Nuclear densitiesafter 14-day BCEC-CM culture (mean nuclear density, 17.0 nuclei/mmBruch's membrane) vs. 21-day BCEC-CM culture (mean nuclear density,27.9) were significantly different (P=0.031). Nuclear densities ofexplants cultured for 14 days in BCEC-CM were significantly higher thanthose of explants cultured for 3 days in BCEC-CM (P<0.05) while 3- vs.7-day and 7- vs. 14-day nuclear densities were not significantlydifferent (P>0.05). There were no statistically significant differencesbetween the control groups cultured for the entire 21-day period inBCEC-CM (P=0.074). Ages between groups were not significantly different(P=0.881: 3-day cohort, mean donor age, 78.3±7.6 yrs.; 7-day cohort,mean donor age, 76.5±6.0 yrs.; 14-day cohort, mean donor age, 77.2±7.1yrs.)

Comparison of RPE Nuclear Density after 21-Day Culture in DifferentMedia and on Different Surfaces

Nuclear densities of fetal RPE after 21-day culture in BCEC-CM or CMvehicle on AMD (including late AMD) and aged, non-AMD explants (currentstudy: CM vehicle, mean donor age, 80.2±7.8 yrs. (data represented inFIG. 28A); BCEC-CM mean donor age, 78.8±7.3 yrs. (combined datarepresented in FIGS. 28A and 21-day controls of FIG. 29)), were comparedwith: 1) 21-day fetal RPE nuclear densities on young explants (meandonor age, 44.8±2.3 yrs.) cultured in RPE medium, 2) aged and early AMDBruch's membrane explants (mean donor age, 73.6±6.4 yrs.) cultured inRPE medium, and 3) BCEC-ECM-resurfaced aged Bruch's membrane (mean donorage, 73.9±7.4 yrs.) cultured in RPE medium (FIG. 30). Of the agedexplants studied, mean donor age of explants cultured in BCEC-CM and CMvehicle (includes eyes with early as well as late AMD) weresignificantly higher than the mean donor age of aged, including earlyAMD, explants cultured in RPE media (P<0.05) but not significantlydifferent from the mean donor age of explants that were resurfaced withBCEC-ECM (aged, non-AMD) (P>0.05). The nuclear densities of fetal RPE onBCEC-ECM-resurfaced aged Bruch's membrane (mean nuclear density, 23.2nuclei/mm Bruch's membrane) and unresurfaced young Bruch's membrane(mean nuclear density, 27.2) were significantly higher than the nucleardensity on aged, untreated Bruch's membrane after culture in RPE medium(mean nuclear density, 11.221) (P<0.05) and were not significantlydifferent from the nuclear density on explants cultured in BCEC-CM (meannuclear density, 23.2) (P>0.05). Nuclear densities of fetal RPE cells inthese three conditions (i.e., BCEC-CM-cultured, BCEC-ECM-resurfaced, andyoung Bruch's membrane) were significantly higher than the nucleardensity on aged and early AMD Bruch's membrane cultured in RPE medium(mean nuclear density, 2.2) (P<0.05). Nuclear densities of cellscultured in RPE medium on aged and early AMD Bruch's membrane weresignificantly higher than nuclear densities of cells cultured in CMvehicle on aged and AMD Bruch's membrane (P<0.05).

ECM Deposition Following Culture in BCEC-CM

Providing a newly-deposited ECM on the surface of Bruch's membrane cansignificantly improve cell survival in long-term organ culture, and theresulting nuclear densities are similar to those observed after culturein BCEC-CM (FIG. 30). To determine whether incubation in BCEC-CMstimulates ECM deposition, which might account for the cell-preservingeffect of BCEC-CM, it was investigated whether RPE ECM deposition onBruch's membrane was increased after culture in BCEC-CM. Since littleRPE cell survival on Bruch's membrane was seen in CM vehicle, assayswere performed to compare ECM deposition after culture in BCEC-CM withECM deposition after culture in standard RPE culture medium where somedegree of RPE resurfacing was more likely (see FIG. 30). To assess ECMdeposition on a non-toxic substrate, ECM deposition onto tissue culturedishes was examined at days-7, -14, and -21 (N=3).

Stained fibers were present on the surface of culture dishes in bothmedia at all three time points. As the time in culture increased, theamount of ECM deposition increased. ECM visualized by Ponceau S stainingshowed deposition after BCEC-CM culture to be more extensive thandeposition after RPE medium culture at all time points (FIG. 31). InBCEC-CM cultures, collagen IV and laminin were present as a thickcoating with defects that became smaller with time in culture (FIGS.32-34). Collagen IV deposition appeared to be more extensive thanlaminin and fibronectin deposition at day-7. At day-14 and -21, collagenIV and laminin showed more uniform coating of the culture dish surfacecompared to day-7. Collagen IV and laminin appeared to be extensivelyco-localized at all three time points (FIGS. 32-34, C). Fibronectinlabeling was on a network of thin fibers at week-1 with diffuse labelingof the culture dish between fibers seen at day-14 and -21. Somecolocalization of fibronectin with laminin was seen (FIGS. 32-34, F),but it was not as extensively co-localized as laminin was with collagenIV. In RPE medium cultures (FIGS. 32-34, G-L), collagen IV and lamininlabeling at day-7 was not as extensive as that seen in BCEC-CM cultureswith labeling seen as an open mesh with localized areas coating theculture dish between the fibers of the mesh. Localized areas of surfacescoated with collagen IV and laminin were more extensive at day-14 and-21 compared to day-7, but there was little, if any, increase inlabeling between day-14 and -21. Faint fibronectin labeling was detectedin RPE medium cultures at all time points with sparse to moderatelabeling seen at day-21. Similar to that observed in BCECCM cultures,collagen IV and laminin showed extensive co-localization. Fibronectinco-localization was difficult to determine due to sparse labeling insome cultures, but it did seem to partially co-localize with laminin(see FIG. 32L). Controls showed diffuse, faint fluorescence withrhodamine filters with secondary antibody only in the 3-week RPE mediumcultures. No detectable fluorescence was found with FITC or rhodaminefilters in preparations at other time points or in CM vehicle culturesat all time points (not shown). Controls in which the antibody wasomitted showed no fluorescence with either filter set (not shown).Calcein imaging confirmed the presence of RPE on Bruch's membraneexplants cultured in both media for 21 days with more extensiveresurfacing and smaller, more compact cells on explants cultured inBCEC-CM (FIGS. 35A and L). On explants cultured in BCEC-CM, SEM andconfocal evaluation showed extensive ECM on the surface of the innercollagenous layer for all three markers (N=6; mean donor age, 79.2±3.17yrs) (FIGS. 35A-F and H-J). The extent and complexity of the ECM variedwithin and between BCEC-CM-treated explants, ranging from a complex meshof thick and thin fibers to a fairly continuous ECM sheet with a roughsurface (FIGS. 35A-F and H-J). Collagen IV and laminin labeling werefound as thick and thin fibers with some localized thickening giving thelabeling a punctate appearance. Both collagen IV and laminin formed acontinuous sheet in localized areas, visualized between fibers. CollagenIV labeling was more variable than that of fibronectin and laminin,sometimes not as extensive. There was some co-localization of collagenIV with laminin. Fibronectin was found mainly in fibers and did notappear to co-localize with laminin. On explants cultured in RPE medium,no or sparse localized labeling was observed despite RPE presence onBruch's membrane as visualized by Calcein imaging (FIGS. 35 L and M).When present, laminin labeling could be seen on short strands. Ifpresent, fibronectin was present in short (thin) strands; sometimeslabeling was punctate. Collagen IV labeling generally was sparse or notpresent. ECM tended to be more prevalent outside the submacular area onthe periphery of the explant. SEM evaluation of the explants showedpredominantly bare ICL or a few strands on the surface of the bare ICLin explants cultured in RPE medium (FIG. 35M). Control explant pieces(omission of primary antibody or both primary and secondary antibodies)showed slight autofluorescence of the tissue (FIGS. 35G and K) at themicroscope settings used to obtain immunolabeling images.

In previous studies examining fetal RPE attachment to aged and AMDsubmacular Bruch's membrane, it was showed that RPE can attach to a highdegree to the RPE basement membrane and to the inner collagenous layer,indicating that attachment to these layers may not be the limitingfactor in cell transplant success. Immunochemistry studies showed thatat days-3 and -7 after seeding, fetal RPE are present on Bruch'smembrane and appear to be fairly healthy based on the appearance ofstained nuclei. Between days-7 and -14, a high degree of cell death hasoccurred, and additional cell death was observed at days-14 and -21. Atthese later time points, abundant condensed and fragmented nuclei arepresent. These studies provide evidence that a method to ensure cellsurvival (vs. attachment alone) must be developed for RPE cellreplacement therapy to be successful in AMD patients.

Previously, it was demonstrated that cell survival on aged submacularBruch's membrane can be enhanced greatly by resurfacing Bruch's membranewith a cell-deposited ECM. This resurfacing strategy was chosen becauseECM deposited by BCECs supports rapid fetal RPE attachment and growth incell culture. It was showed that resurfacing aged Bruch's membrane withBCEC-ECM improved long-term cell survival significantly (>200%). Alimitation to the feasibility of utilizing this ECM in clinicalapplications was the insolubility of the ECM and the resulting low yieldof proteins with ECM harvest, both for transfer to Bruch's membrane andfor quantitative analysis. In the present study, BCEC-CM was harvestedfrom BCECs cultured under conditions similar to those that yieldBCEC-ECM-coated culture dishes and BCEC-ECM deposition on Bruch'smembrane. The rationale for this choice was based on the reportedabundance of potentially cell-supporting proteins secreted into themedium, such as ECM and ECM-associated molecules. In the present study,it was demonstrated that cell survival is greatly enhanced when RPEcells are exposed to this BCEC-CM in long-term culture and, importantly,that cell survival is enhanced on submacular Bruch's membrane of AMDeyes. It was showed previously that RPE survival on AMD submacularBruch's membrane explants was severely impaired after culture in RPEmedium. However, the nuclear density of fetal RPE on submacular humanBruch's membrane cultured in RPE medium is significantly higher comparedto the nuclear density of fetal RPE cultured in CM vehicle (FIG. 30).This difference could be related to the significantly lower mean donorage of the explants cultured in RPE media and, therefore, possibly feweraged and AMD changes to Bruch's membrane. However, culture on non-AMDand AMD explants was similarly poor in CM vehicle. It seems likely thatthe serum in the RPE medium aided in supporting cell survival to aslight degree but not to the degree seen in BCEC-CM.

BCEC-CM may supply soluble matrix proteins for ECM deposition, stimulateECM deposition, and/or stimulate the RPE in some other fashion, thusallowing better survival that could lead to increased ECM deposition onaged and AMD submacular Bruch's membrane. The presence of increased ECMdeposition under the cells cultured in BCEC-CM compared to cellscultured in RPE medium may reveal a mechanism by which cell survival isenhanced, perhaps in the same manner that BCEC-ECM resurfaced explantssupport long-term cell survival on submacular human Bruch's membrane. Itis not known if the ECM deposition per se enabled cell survival in thesame way as resurfacing Bruch's membrane with BCEC-ECM or if the ECMdeposition was a reflection of long-term survival of the cells byanother mechanism. When RPE cell survival is observed on explantscultured in RPE medium, the cells are not as differentiated as thosecultured in BCECCM and have not deposited ECM to any degree. The degreeof ECM deposition by RPE on culture dishes in RPE medium or in BCEC-CMis much greater than that observed on Bruch's membrane explants. Thisdifference may arise because RPE appear to mature more slowly on Bruch'smembrane, and a certain degree of maturity must be obtained before ECMdeposition can occur. Differences in the amount and composition of ECMdeposition may also be related to differences in cellular response tothe underlying substrate. Since the antibodies used in this study werenot human-specific, it was not known if deposited proteins originatedfrom the BCEC-CM (bovine proteins) or from protein synthesis by the RPEor both. As mentioned previously, when cultured in standard RPE medium,fetal RPE can survive to a high degree up to 7 days in organ culture onsubmacular human Bruch's membrane. After day-7, survival is impairedwith decreasing presence of cells as time in culture increases further.The necessity of long-term presence of BCEC-CM to assure cell survivalis consistent with the notion that sustained cell stimulation is afactor in assuring cell survival. The RPE survival after culture inBCEC-CM as measured by nuclear density was statistically similar to thatof cells cultured on BCEC-ECM and also similar to that observed on youngBruch's membrane, the latter both cultured in RPE medium (FIG. 30). Thenuclear densities observed in these studies are much lower than thenuclear densities of submacular in situ RPE even when comparing agematched nuclear densities and also much lower than the nuclear densitiesof fetal RPE in culture. The lower nuclear density on submacular Bruch'smembrane after culture in BCEC-CM is partly due to the existence ofdefects in surface coverage on some explants. Many explants are almostfully resurfaced or are fully resurfaced after culture in BCEC-CM, andmany cells appear to show some morphological features of differentiation(e.g., apical processes, tight junctions). However, there is variabilityin cell size with some cells fairly large, especially compared to thesize of cultured fetal RPE. One source of the variability in cellbehavior between explants might be biological variability in thecomposition of BCEC-CM between batches, as some batches appeared to beless effective than others, showing more and larger defects in surfacecoverage and larger and flatter cells. Another source of variability incell behavior arises from differences in cell survival on localizedareas of Bruch's membrane within explants, as some explants showingexcellent overall resurfacing also exhibited small localized defects inRPE coverage (e.g., FIGS. 24 and 26). Lastly, particularly in referenceto the AMD cohort, variability in cell behavior on explants with CNVs islikely related, at least in part, to the differences in the Bruch'smembrane surface following CNV removal since no additional debridementwas performed. The degree of damage to Bruch's membrane orpreservation/removal of deposits on Bruch's membrane following CNVremoval were highly variable within and between donor eyes. In aprevious study, hES-RPE were shown to have the potential to survive onequatorial and submacular Bruch's membrane to a similar degree as fetalRPE after day-21 culture in RPE medium. On submacular Bruch's membrane,the survival was poor for both cell types with hES-RPE survival impairedat earlier times in culture than fetal RPE. In these previous studies,hES-RPE was from frozen stock, which is a possible cause for thedifference in initial cell survival since the fetal RPE were from freshcultures. However, in the current study, hES-RPE were from fresh stock,and although the nuclear densities of hES-RPE cultured in BCEC-CM weresimilar to those of fetal RPE, hES-RPE in general were very flat and notdifferentiated to the same degree as fetal RPE on submacular Bruch'smembrane. These results imply that hES-RPE may take longer to acquiremature RPE features on Bruch's membrane compared to fetal RPE,consistent with behavior observed in cell culture. Whether fetal RPE orhES-RPE can achieve size and differentiation features found in cellculture or in situ and whether the cells can perform RPE functions arefuture studies that must be considered.

Cultured aged adult RPE were generally larger than hES-RPE and fetal RPEin cell culture and on Bruch's membrane. On Bruch's membrane, most adultRPE showed well developed apical processes even in very large flatcells, but their survival in general was not as good as that of fetalRPE or hES-RPE. The nuclear density of cultured adult RPE after BCEC-CMculture (10.0±0.95) was not significantly different (P=0.887) from thenuclear density of fetal RPE after RPE medium culture (11.2±1.7, FIG.30). These results indicate that while culture in BCEC-CM significantlyenhances RPE cell survival on aged Bruch's membrane, aged adult RPE maynot be the best choice for cell transplantation especially when comparedto the resurfacing achieved by fetal RPE and hES-RPE.

There is no other technique known that promotes such robust RPE survivalon submacular AMD Bruch's membrane (including eyes with geographicatrophy and CNVs). 19, 20 Identification of the critical components ofthis BCEC-CM and RPE function testing are the next steps in thedevelopment of a surgically usable adjunct to improve RPE survival anddifferentiation on submacular human AMD Bruch's membrane.

TABLE 9 Donor information SI Table 1. Donor information SubmacularPathology Donor Age Ethnicity/sex CM cultured CM Vehicle cultured A.hES-RPE on AMD and non-AMD Bruch's membrane (mean age 80.17 ± 3.41 yrs.)68 CF Few mixed size drusen Normal 74 CF Confluent soft drusen Softdrusen 80 CM Normal Normal 83 CM RPE hyperpigmentation, mixed size RPEhyperpigmentation, mixed drusen size drusen 84 CF Normal Normal 92 CFConfluent soft drusen Confluent soft drusen B. Fetal RPE on non-AMDBruch's membrane (mean age 77.8 ± 1.69 yrs.) 67 CM Few mixed size drusenFew Hard drusen 75 CM Focal RPE hyperpigmentation Normal 76 CM NormalNormal 77 CM Normal Normal 78 CM Normal Normal 79 CM Few hard drusen Fewhard drusen 81 CM Few hard drusen Normal 83 CM Few hard drusen Few harddrusen 84 CF Few mixed size drusen Few mixed size drusen C. Fetal RPE onAMD Bruch's membrane (mean age 81.9 ± 2.49 yrs) 62 CM Soft drusen Softdrusen 75 CF Large CNV Large CNV 76 CF Confluent soft drusen Confluentsoft drusen 79 CF CNV, focal RPE hyperpigmentation, focal RPEhyperpigmentation soft drusen 79 CF CNV Few hard drusen, focal RPEhyperpigmentation 79 CF Large CNV CNV 81 CF CNV, (Fuch’s dystrophydonor) Mixed size drusen 82 CF Extrafoveal GA, soft drusen ExtrafovealGA, soft drusen 86 CM Extensive soft drusen, small CNV Extensive softdrusen, small CNV 86 CF CNV CNV 90 CM Large CNV Large CNV 92 CF GA withcentral RPE preservation GA with large, calcified drusen 98 CM Confluentsoft drusen Confluent soft drusen D. Adult RPE on AMD and non-AMDBruch's membrane (mean age 75.7 ± 1.38 yrs.) 71 CF Extrafoveal GA, softdrusen Unknown 73 CF Unknown (poor RPE preservation) Unknown 73 CMNormal Normal 75 CF Normal Few hard drusen 78 CF Normal Normal 80 CMCluster of soft drusen Few intermediate size drusen 80 CM Extrafovealdrusen including calcified Few hard drusen drusen, macula normal Therewas no statistically significant difference in ages of Bruch's membranebetween groups (hES-RPE vs. combined AMD and non-AMD fRPE vs. adult RPE,One Way ANOVA P = 0.354). For AMD vs non-AMD, normal, there was nosignificant difference in the ages of the two groups (unpaired t-test, P= 0.226).

All publications cited in the specification, both patent publicationsand non-patent publications, are indicative of the level of skill ofthose skilled in the art to which this invention pertains. All thesepublications are herein fully incorporated by reference to the sameextent as if each individual publication were specifically andindividually indicated as being incorporated by reference.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the following claims.

1. A method of increasing survival and/or differentiation of targetcells on a base matrix, the method comprising: creating a cell-madeextracellular matrix on said base matrix to produce a modified basematrix and administering said target cells to said modified base matrix.2. The method of claim 1, wherein the step of creating the cell-madeextracellular matrix on said base matrix to produce the modified basematrix is performed in vitro.
 3. The method of claim 1, wherein the stepof administering said target cells to said modified base matrix isperformed in vivo.
 4. The method of claim 1, wherein the step ofcreating the cell-made extracellular matrix on said base matrix toproduce the modified base matrix is performed in vivo.
 5. A modifiedbase matrix for survival and/or differentiation of target cells thereon,the modified base matrix comprising a cell-made extracellular matrixthereon.
 6. The modified base matrix of claim 5, wherein the modifiedbase matrix is produced by culturing cells capable of forming saidcell-made extracellular matrix on the base matrix.
 7. The modified basematrix of claim 5, wherein said modified base matrix is produced bytreating the base matrix with at least an active fraction of aconditioned media from culturing cells capable of forming said cell-madeextracellular matrix on the base matrix.
 8. The modified matrix of claim5, wherein said base matrix is Bruch's membrane.
 9. The modified basematrix of claim 8, wherein the Bruch's membrane is undergoing maculardegeneration.
 10. The modified matrix of claim 5, wherein said basematrix is a synthetic polymer-based matrix.
 11. The modified base matrixof claim 10, wherein the synthetic polymer is selected from polylacticacid (PLA), polyglycolic acid (PGA), poly(lactide-co-glycolide) (PLGA),poly(methyl methacrylate) (PMMA), polyorthoesters, and any combinationsthereof.
 12. The modified base matrix of claim 11, wherein the syntheticpolymer is polycaprolactone (PCL).
 13. The modified base matrix of claim5, wherein said target cells are selected from RPE, umbilical cells,placental cells, adult stem cells, embryonic stem cells, fetal RPE,adult iris pigment epithelial (IPE) cells, bone marrow-derived stemcells, Schwann cells, neural progenitor cells, and any combinationthereof.
 14. The modified base matrix of claim 5, wherein said targetcells are autologous.
 15. The modified base matrix of claim 5, whereinsaid target cells are fetal RPEs.
 16. The modified base matrix of claim5, wherein the target cells are adult or embryonic stem cells or aredifferentiated from adult or embryonic stem cells.
 17. A conditionedmedia from culturing cells capable of forming a cell-made extracellularmatrix on the base matrix of claim
 5. 18. The conditioned media of claim17, which is serum-free.
 19. The conditioned media of claim 17, whereinsaid cells capable of forming said cell-made extracellular matrix on thebase matrix are selected from corneal endothelial cells, RPE cells, IPEcells, embryonic stem cells, bone marrow-derived stem cells, placentalcells, and/or umbilical cells.
 20. The conditioned media of claim 19,wherein said cells are corneal endothelial cells.
 21. The conditionedmedia of claim 19, wherein the conditioned media or at least a fractionthereof is produced by culturing cells capable of forming said cell-madeextracellular matrix.
 22. An active fraction of a conditioned media ofclaim 17, characterized by a depletion of biologically active componentshaving MW of less than about 100 kD.
 23. The active fraction of claim22, wherein the active fraction of the conditioned media from culturingcells capable of forming said cell-made extracellular matrix on the basematrix is formed by a depletion of biologically active components havingMW of less than about 100 kD.
 24. A kit for promoting the survivaland/or differentiation of target cells on a base matrix, comprising: a)a set of instructions and at least one of: b) an efficient amount ofcells capable of forming a cell-made extracellular matrix; and c) atleast an active fraction of a conditioned media or an extracellularmatrix from the cells capable of forming a cell-made extracellularmatrix.
 25. The kit of claim 24, wherein the cells capable of formingthe cell base and at least the active fraction of the conditioned mediaor the extracellular matrix from the cells capable of forming thecell-made extracellular matrix are according claim
 5. 26. A kit forpromoting survival and differentiation of target cells comprising a setof instructions and a modified base matrix according to claim
 5. 27. Thekit of claim 24 further comprising an effective amount of target cellsselected from RPE, umbilical cells, placental cells, adult stem cells,cells differentiated from adult stem cells, ES cells, cellsdifferentiated from ES cells, bone marrow-derived stem cells, fetal RPE,adult iris pigment epithelial (IPE) cells, Schwann cells, and anycombination thereof, derived from autologous or allogenic source.
 28. Amethod for making a conditioned medium, comprising obtaining a pluralityof cells capable of forming a cell-made extracellular matrix; culturingthe cells in a first medium for a period of time to form a secondmedium; and collecting the second medium thereby making the conditionedmedium.
 29. The method of claim 28, wherein the first medium is free ofserum.
 30. The method of claim 28, wherein the cells are selected fromthe group consisting of corneal endothelial cells, RPE cells, IPE cells,embryonic stem cells, bone marrow-derived stem cells, placental cells,and umbilical cells.
 31. The method of claim 30 wherein the cells arecorneal endothelial cells.
 32. The method of claim 28, wherein theperiod of time is 1-5 days.
 33. The method of claim 32, wherein theperiod of time is 2-4 days.
 34. The method of claim 33, wherein theperiod of time is 3 days.
 35. A composition comprising a fraction of aconditioned medium of claim
 17. 36. The composition of claim 35, whereinthe fraction comprises molecules having MW of less than 3 kD.
 37. Thecomposition of claim 35, wherein the fraction comprises molecules havingMW of less than 50 kD.
 38. The composition of claim 37, wherein thefraction comprises molecules having MW of 10-50 kD.
 39. The compositionof claim 38, wherein the fraction comprises molecules having MW of 30-50kD.
 40. The composition of claim 35, comprising a first fraction and asecond fraction of the medium, wherein (i) the first fraction comprisesmolecules having MW of less than 3 kD, and (ii) the second fractioncomprises molecules having MW of 10-50 kD.
 41. The composition of claim35, wherein the fraction comprises one or more proteins selected fromthose in Table
 6. 42. The composition of claim 35, wherein thecomposition is a pharmaceutical composition and comprises apharmaceutically acceptable carrier.
 43. The composition of claim 35,wherein the composition is a cell culture medium for use in storing,preserving, or inducing differentiation of cells or tissues.
 44. Amethod for treating age-related macular degeneration (AMD), comprisingidentifying a subject in need thereof; and administering to the subjectan effective amount of the composition of claim
 35. 45. A packagingproduct comprising a composition of claim 44; a cell or a piece oftissue, and a container holding the composition.
 46. The packagingproduct of claim 45, wherein the cell is selected from the groupconsisting of retinal pigment epithelial (RPE) cells, stem cells, andcorneal cells.
 47. The packaging product of claim 45, wherein the tissueis selected from the group consisting of RPE derived from precursorcells, RPE derived from human embryonic stem cells, RPE derived fromiPSC stem cells, whole retinae, whole cornea, tissues, and neuraltissues and organs.
 48. The packaging product of claim 45, wherein thecell is in suspension or in a support matrix designed for cell delivery.49. The packaging product of claim 45, where in the composition has atemperature within the range of 0-40° C.
 50. The packaging product ofclaim 49, wherein the composition has a temperature within the range of4-30° C.
 51. The packaging product of claim 50, wherein the compositionhas a temperature within the range of 15-25° C.
 52. The packagingproduct of claim 44, wherein the container is sealed.